Method of inducing differentiation from pluripotent stem cells to germ cells

ABSTRACT

This invention provides a method of producing a primordial germ cell-like cell (PGCLC) from an epiblast isolated from an embryo or an epiblast-like cell (EpiLC) induced from a pluripotent stem cell (PSC), which comprises allowing the epiblast or EpiLC to express exogenous transcription factor(s) selected from the group consisting of: (i) Blimp1, Prdm14 and Tfap2c; ii) Blimp1 and Prdm14; (iii) Blimp1 and Tfap2c; (iv) Prdm14 and Tfap2c; and (v) Prdm14; thereby inducing the epiblast or EpiLC into a PGC state without acquiring transient mesodermal program.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is the U.S. national phase of International Patent Application No. PCT/JP2014/055888, filed Feb. 28, 2014, which claims the benefit of U.S. Provisional Patent Application No. 61/771,619, filed on Mar. 1, 2013, which are incorporated by reference in their entireties herein.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: 113,811 bytes ASCII (Text) file named 721764CorrectedSequenceListing created Aug. 31, 2017.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a method of inducing primordial germ cell-like cells from epiblast-like cells using transcription factor(s), a reagent therefor containing the transcription factor(s), a cell population containing primordial germ cell-like cells obtained from the method and a method of inducing cells belonging to the germ cell lineage from the cell population.

BACKGROUND OF THE INVENTION

The germ cell lineage ensures the continuity of life through the generation of male and female gametes, which unite to form a totipotent zygote. Germ cell specification and development are vital in reproduction and heredity. In mice, primordial germ cells (PGCs), precursors both for spermatozoa and oocytes, arise in the epiblasts in response to cytokines, including, most importantly, bone morphogenetic protein 4 (BMP4), from extraembryonic tissues (non-patent documents 1 and 2). We have recently established a culture system to induce embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) into epiblast-like cells (EpiLCs) using cytokines including activin A (ActA) and basic fibroblast growth factor (bFGF), and then into primordial germ cell-like cells (PGCLCs) using cytokines including BMP4 (patent document 1, non-patent documents 3 and 4). Male and female PGCLCs bear full potential for spermatogenesis and oogenesis, and thus may have potential for unveiling the mechanism of and regulating the germ-cell specification pathway and subsequent development in vitro (patent document 1, non-patent documents 3 and 4).

In replace of cytokines, forced expression of exogenous transcription factors (TFs) may activate endogenous key transcription circuitry for PGC specification. However, our previous studies revealed that PGC specification involves complex regulation of a large number of genes, while some key TFs were identified (non-patent document 5). Accordingly, the TFs sufficient for the induction of germ cell fate and the precise mechanism of action of key TFs remain unknown.

PRIOR ART REFERENCES CITED Patent Documents

-   1. WO 2012/020687

Non-Patent Documents

-   1. Lawson, K. A. et al. Bmp4 is required for the generation of     primordial germ cells in the mouse embryo. Genes Dev 13, 424-436.     (1999). -   2. Ohinata, Y. et al. A signaling principle for the specification of     the germ cell lineage in mice. Cell 137, 571-584 (2009). -   3. Hayashi, K., Ohta, H., Kurimoto, K., Aramaki, S. & Saitou, M.     Reconstitution of the mouse germ cell specification pathway in     culture by pluripotent stem cells. Cell 146, 519-532 (2011). -   4. Hayashi, K. et al. Offspring from oocytes derived from in vitro     primordial germ cell-like cells in mice. Science 338, 971-975     (2012). -   5. Kurimoto, K. et al. Complex genome-wide transcription dynamics     orchestrated by Blimp1 for the specification of the germ cell     lineage in mice. Genes Dev 22, 1617-1635 (2008).

SUMMARY OF THE INVENTION

Therefore, an object of the present invention is to provide a method of inducing PGCLCs, which are competent for generating normal germ cells and healthy offsprings, from epiblasts of culture equivalents thereof (i.e., EpiLCs) with high efficiency and reproducibility, without cytokines, but using forced expression of exogenous TFs. It is another object of the present invention to provide a reagent for inducing PGCLCs from epiblasts or EpiLCs containing the TFs that allow epiblasts/EpiLCs to activate endogenous key transcription circuitry for PGC specification, or nucleic acids encoding the same.

To this end, we set out to explore TFs whose forced expression may be sufficient to confer the PGC fate onto EpiLCs, based on the system for recapitulating the mammalian germ-cell specification pathway in vitro we reported previously (patent document 1, non-patent documents 3 and 4, supra). We decided to focus on three TFs, Blimp1 (also known as Prdm1), Prdm14 and Tfap2c (also known as AP2γ), since these factors are expressed in epiblasts/EpiLCs stimulated by BMP4 (see for example, FIG. 2b ). First, we derived ESCs expressing mVenus and ECFP under the control of Blimp1 and stella (also known as Dppa3/Pgc7) regulatory elements (BVSC), respectively (Reproduction 136, 503-514 (2008)), and reverse tetracycline transactivator (rtTA) under the control of the constitutively active Rosa26 locus (Cell 121, 465-477 (2005))(BVSCR26rtTA ESCs) (FIG. 1a ). During development, Blimp1 expression signifies the onset of PGC specification, whereas stella begins expression in the established PGCs (Nature 436, 207-213 (2005); Nature 418, 293-300 (2002); Mech Dev 113, 91-94. (2002)), and the BVSC expression is a faithful indicator for PGC specification and development both in vivo and in vitro (Cell 146, 519-532 (2011); Science 338, 971-975 (2012); Reproduction 136, 503-514 (2008)). Next, we constructed piggyBac transposon-based vectors expressing Blimp1, Prdm14 or Tfap2c under the control of tetracycline regulatory elements (TREs) (FIG. 5d ) and infected the BVSCR26rtTA ESCs (XY karyotype) (FIG. 5a-c ) with these vectors to isolate BVSCR26rtTA ESCs bearing transgenes for all three TFs (BVSCR26rtTA BP14A cells), two of the three TFs (BVSCR26rtTA BP14, BA and P14A cells), or one of the three TFs (BVSCR26rtTA B, P14, and A cells) (FIG. 1a , FIG. 5d, e ).

We induced the TF-infected BVSCR26rtTA ESCs into EpiLCs using ActA and bFGF (Cell 146, 519-532 (2011); Science 338, 971-975 (2012)), and then cultured in the absence of cytokines relevant to PGCLC induction (BMP4, LIF, SCF, BMP8b and EGF; Cell 146, 519-532 (2011); Science 338, 971-975 (2012)) with doxycycline (Dox). As a result, simultaneous over-expression of three TFs (Blimp1, Prdm14 and Tfap2c; BP14A) or two of the three TFs (BP14, BA and P14A) directs EpiLCs swiftly and highly efficiently into a PGC state with endogenous transcription circuitry. The induction of the PGC state on EpiLCs minimally requires Prdm14 (P14) but not Blimp1 (B) or Tfap2c (A).

Quantitative PCR (Q-PCR) and global gene expression analyses revealed that the TF-induced PGC state reconstitutes key transcriptome and epigenetic reprogramming in PGCs, but bypasses a mesodermal program that accompanies PGC specification in vivo and in vitro by cytokines including BMP4.

Importantly, the TF-induced PGCLCs robustly contribute to spermatogenesis and fertile offspring.

We conducted further investigations based on these findings, and have developed the present invention.

Accordingly, the present invention provides the following.

[1] A method of producing a primordial germ cell-like cell (PGCLC) from an isolated epiblast or epiblast-like cell (EpiLC), which comprises allowing the epiblast or EpiLC to express exogenous transcription factor(s) selected from the group consisting of:

(i) Blimp1, Prdm14 and Tfap2c;

(ii) Blimp1 and Prdm14;

(iii) Blimp1 and Tfap2c;

(iv) Prdm14 and Tfap2c; and

(v) Prdm14;

thereby inducing the epiblast or EpiLC into a PGC state without acquiring transient mesodermal program.

[2] The method according to [1] above, wherein the exogenous transcription factor(s) or nucleic acid(s) encoding the same is/are introduced into the epiblast or EpiLC.

[3] The method according to [1] above, wherein the nucleic acid(s) encoding the exogenous transcription factor(s) has/have been introduced into the epiblast or EpiLC, in a form capable of being conditionally expressed, prior to the induction of the epiblast or EpiLC. [4] The method according to [3] above, wherein the epiblast or EpiLC is cultured under conditions which the nucleic acid(s) encoding the exogenous transcription factor(s) is/are expressed for 1 to 5 days. [5] The method according to any one of [1] to [4] above, wherein the EpiLC is obtained by culturing a pluripotent stem cell (PSC) in the presence of activin A (ActA), optionally in the presence of further basic fibroblast growth factor (bFGF) and/or Knockout™ Serum Replacement (KSR). [6] The method according to [5] above, wherein the PSC is an embryonic stem cell (ESC) or induced pluripotent stem cell (iPSC). [7] The method according to any one of [1] to [6] above, wherein the nucleic acid(s) encoding the exogenous transcription factor(s) is in a form capable of disappearing from the PGCLC. [8] The method according to [7] above, wherein the nucleic acid(s) is/are carried on vector(s) selected from the group consisting of plasmid, episomal vector, transposon, adenoviral vector and Sendai viral vector. [9] The method according to any one of [1] to [8] above, wherein the EpiLC is derived from mouse or human. [10] A reagent for inducing an isolated epiblast or EpiLC into a PGCLC comprising transcription factor(s) selected from the group consisting of: (i) Blimp1, Prdm14 and Tfap2c; (ii) Blimp1 and Prdm14; (iii) Blimp1 and Tfap2c; (iv) Prdm14 and Tfap2c; (v) Prdm14; or nucleic acid(s) encoding the transcription factor(s). [11] The reagent according to [10] above, wherein the nucleic acid(s) encoding the transcription factor(s) is/are in a form capable of being conditionally expressed in the epiblast or EpiLC. [12] An isolated epiblast or EpiLC comprising nucleic acid(s) encoding exogenous transcription factor(s) selected from the group consisting of: (i) Blimp1, Prdm14 and Tfap2c; (ii) Blimp1 and Prdm14; (iii) Blimp1 and Tfap2c; (iv) Prdm14 and Tfap2c; and (v) Prdm14; wherein the nucleic acid(s) is/are in a form capable of being conditionally expressed in the epiblast or EpiLC. [13] An isolated PSC comprising nucleic acid(s) encoding exogenous transcription factor(s) selected from the group consisting of: (i) Blimp1, Prdm14 and Tfap2c; (ii) Blimp1 and Prdm14; (iii) Blimp1 and Tfap2c; (iv) Prdm14 and Tfap2c; and (v) Prdm14; wherein the nucleic acid(s) is/are in a form capable of being conditionally expressed in an EpiLC differentiated from the PSC. [14] A kit for inducing an isolated epiblast or EpiLC into a PGCLC comprising the epiblast or EpiLC according to [12] above; and a reagent that allows the epiblast or EpiLC to express the exogenous transcription factor(s). [15] A kit for inducing an isolated PSC into a PGCLC comprising the PSC according to [13] above; a reagent for inducing the PSC into an EpiLC comprising ActA and optionally bFGF and/or KSR; and a reagent that allows the EpiLC to express the exogenous transcription factor(s). [16] A method of producing a PGCLC from a PSC, which comprises the following steps I) and II): I) the step for producing an EpiLC by culturing a PSC in the presence of ActA, optionally in the presence of further bFGF and/or KSR; II) the step for inducing the EpiLC obtained in the step I) into a PGCLC by the method according to any one of [1] to [9] above. [17] The method according to [16] above, which further comprises: III) the step for selecting a Blimp1-positive cell from the cells obtained in the step II). [18] A method of producing a variety of cell types derived from epiblast which comprises utilizing the PGCLC cell population obtained by the method according to [16] or [17] above as a cell source.

Here we show that, without cytokines including BMP4, simultaneous over-expression of three TFs, Blimp1, Prdm14 and Tfap2c, directs EpiLCs swiftly and highly efficiently into a PGC state with endogenous transcription circuitry. Furthermore, the TF-induced PGCLCs robustly contribute to spermatogenesis and fertile offspring. In view of clinical application (e.g., fertility treatment), it is highly desirable to prepare germ cells under serum-free and xeno-free conditions. Since human recombinant cytokines are expensive, the present invention contributes to reduce the cost of germ cell production for human therapy.

Our findings provide not only a novel insight into the transcriptional logic that creates a germ cell state, but also a foundation for the TF-based reconstitution and regulation of mammalian gametogenesis.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 shows an induction of a PGC-like state by transcription factors. FIG. 1a , Scheme for the BVSCR26rtTA cells and the piggyBac transposon-based vectors with tetracycline-responsive promoters driving transcription factor and β-geo expression. FIG. 1b , Induction of BVSC with (top) or without (bottom) Dox (1.5 μg/ml) in floating aggregates of EpiLCs induced from BVSCR26rtTA BP14A cells during the 6-day observation period. Bar, 200 μm. FIG. 1c , FACS analysis of BVSC induction by Dox (1.5 μg/ml) in floating aggregates of EpiLCs induced from BVSCR26rtTA BP14A cells (Line 3˜3) (top) or parental BVSCR26rtTA cells (bottom). FIG. 1d , Expression of the indicated genes measured by Q-PCR in TF (BP14A)- or cytokine-induced BV-positive cells (red and orange, respectively) or in the whole aggregates of EpiLCs from parental BVSCR26rtTA cells with Dox (grey) during the 4-day observation period. For each gene, the ΔCt from the average Ct values of two independent housekeeping genes, Arbp and Ppia, is shown on the log 2 scale. For each gene, mean value of two independent experiments with two technical replicates is shown. FIG. 1e , Efficiencies of BVSC induction at d2 plotted against whole exogenous TF transcript levels 12 hrs after induction in a number of independent clones of the indicated cells.

FIG. 2 shows an induction of a PGC-like state by TFs does not involve BMP4 signalling but requires an appropriate epigenetic background. FIG. 2a , Induction of BVSC at day 2 by Dox (1.5 μg/ml) in BVSCR26rtTA BP14A (Line 3-3)-derived EpiLCs or by BMP4 (500 ng/ml) in parental BVSCR26rtTA-derived EpiLCs without or with an inhibitor for ALK2/3, LDN193189 (120 pM). Negative controls without induction are shown on the right. Bar, 200 μm. FIG. 2b , Expression of the indicated genes at day 2 in whole EpiLC aggregates induced by Dox (BVSCR26rtTA BP14A cells) or BMP4 (BVSCR26rtTA cells) without or with LDN193189. For each gene, the ΔCt from the average Ct values of two independent housekeeping genes, Arbp and Ppia, is shown on the log 2 scale. FIG. 2c , Induction of BVSC at day 2 with or without Dox in floating aggregates of BVSCR26rtTA BP14A ESCs (top) or EpiLCs (bottom). Bar, 200 μm. FIG. 2d , FACS analysis of BVSC induction in the cells shown in FIG. 2 c.

FIG. 3 shows global transcription profiles during PGCLC induction by TFs or by cytokines and epigenetic properties of TF-PGCLCs. FIG. 3a , PCA of the indicated cells. FIG. 3b , Immunofluorescence analysis of H3K9me2 (top) or H3K27me3 (bottom) levels (red) in d4 BV-positive TF (BP14A)-PGCLCs (GFP-positive, delineated by dotted lines, Line 3-3) compared to those in EpiLCs (DNMT3B-positive, Line 3˜3). DAPI staining is shown on the left. Bar, 20 μm. FIG. 3c , Bisulfite sequence analysis of methylated cytosine in the differentially methylated regions (DMRs) of the imprinted genes in EpiLCs and d4 BV-positive TF (BP14A, Line 3-3)-PGCLCs. White and black circles represent unmethylated and methylated cytosines.

FIG. 4 shows a spermatogenesis and fertile offspring from TF (BP14A)-PGCLCs. FIG. 4a , FACS of BV-positive TF (BP14A, Line 3-10)-PGCLCs for injection into seminiferous tubules. FIG. 4b , Seminiferous tubules of a W/W^(v) mouse injected by TF-PGCLCs showing spermatogenesis. An arrow indicates an empty tubule. Bar, 500 μm. FIG. 4c , Hematoxylin-eosin staining of a section of a W/W^(v) mouse testis injected with TF-PGCLCs showing apparently normal spermatogenesis. The asterisk indicates an empty tubule. Bar, 50 μm. FIG. 4d , Spermatozoa (arrows) from TF-PGCLCs. Bar, 50 μm. FIG. 4e , Zygotes at pronuclear stages generated by injection of TF-PGCLC-derived sperm into wild-type oocytes by ICSI. Bar, 50 μm. FIG. 4 f, 2-cell embryos from zygotes in FIG. 4e . Bar, 50 μm. FIGS. 4g and 4h , Apparently normal offspring (g, h) and placenta (g) derived from TF-PGCLC-derived sperm. FIG. 4i , Genotyping of the offspring from TF-PGCLC-derived sperm by exogenous TFs, BVSC, and R26rtTA. FIG. 4j , A fertile female derived from TF-PGCLC-derived sperm. FIG. 4k , Spermatogenesis by TF (BP14A)- or Ck-PGCLCs.

FIG. 5 shows generation of BVSCR26rtTA ESCs expressing key transcription factors under the control of tetracycline. FIG. 5 a, Chimeras generated by parental BVSCR26rtTA ESCs. FIG. 5b , Rate for the birth of chimeric offspring with parental BVSCR26rtTA ESCs. FIG. 5c , Germline transmission in chimeras of parental BVSCR26rtTA ESCs. FIG. 5d , Schematic representation of the piggyBac transposon-based vector bearing a tetracycline responsive promoter driving the expression of a cloned gene together with β-geo. Blimp1, Prdm14, and Tfap2c were cloned between attB1 and attB2 and are expressed together with β-geo through IRES (internal ribosomal entry sequence). FIG. 5e , Southern blot analysis for the estimation of the copy number of the integrated piggyback transposon vector in each BVSCR26rtTA transfectant. A fragment of β-geo downstream of the BamHI site indicated by the black bar was used as a probe. The estimated copy number for each transfectant is indicated at the bottom.

FIG. 6 shows a Dox-dose-dependent induction of BVSC in floating aggregates of EpiLCs induced from BVSCR26rtTA BP14A ESCs. FIG. 6a , Scheme for the induction of key TFs by Dox in floating aggregates of EpiLCs induced from ESCs. Since the parental BVSCR26rtTA ESC-derived EpiLCs induced for 36 hrs exhibited the most efficient PGCLC induction by cytokines, we consistently used EpiLCs induced for 36 hrs from the transfectants of the BVSCR26rtTA ESCs for the induction of key TFs by Dox. FIG. 6b , Expression levels of the indicated genes in BV-positive cells of the floating aggregates of EpiLCs from BVSCR26rtTA BP14A cells (Line 3-3) induced by Dox for 12 hrs. For each gene, the ΔCT from the average CT values of two independent housekeeping genes, Arbp and Ppia, is shown on the log 2 scale. For each gene, mean value of two independent experiments with two technical replicates is shown. See also FIG. 8. FIG. 6c , β-galactosidase staining of dissociated single cells of floating aggregates of EpiLCs from BVSCR26rtTA BP14A cells with or without Dox for 12 hrs. Bar, 20 μm. See also FIG. 8. FIG. 6d , Dox-dose-dependent induction of BVSC in floating aggregates of EpiLCs induced from BVSCR26rtTA BP14A ESCs (Line 3-10). Bright field (BF) images and immunofluorescence images for BV and SC of floating aggregates of EpiLCs treated with the indicated concentrations of Dox (μg/ml) or the cytokines [BMP4 (500 ng/ml), LIF (1000 U/ml), BMP8A (500 ng/ml), SCF (100 ng/ml), EGF (50 ng/ml)] for 2 days are shown. Bar, 200 μm. FIG. 6e , Percentage of BV- and BVSC-positive cells determined by FACS in the floating aggregates of EpiLCs shown in FIG. 6d . FIG. 6f , Cytokine-induced BVSC expression during the 6-day period in floating aggregates of EpiLCs from parental BVSCR26rtTA ESCs. Bar, 200 μm.

FIG. 7 shows an induction of a PGC-like state by one or two TFs. FIG. 7a , Induction of BVSC at day 2 with (left) or without (right) Dox (1.5 μg/ml) in floating aggregates of EpiLCs induced from BVSCR26rtTA BP14A, BP14, BA, P14A, B, P14, and A cells and from the parental BVSCR26rtTA cells. Results of three independent clones from each cell type are shown. Bar, 200 μm. FIG. 7b , Induction of BVSC at day 2 and 4 analyzed on FACS in floating aggregates of EpiLCs induced from BVSCR26rtTA BP14, BA, P14A, B, P14, and A cells. The clone numbers used for the analysis are shown in parentheses. FIG. 7c , Percentage of BVSC-positive cells determined by FACS in floating aggregates of EpiLCs shown in FIG. 7a . Mean values with value ranges from at least two independent experiments for each line are shown. FIG. 7d , Expression of the indicated genes measured by Q-PCR in TF (BP14A, BP14, BA, P14A, P14)- or cytokine-induced BV-positive cells or in the whole aggregates of EpiLCs from parental BVSCR26rtTA cells with Dox during the 4-day period. For each gene, the ΔCT from the average CT values of two independent housekeeping genes, Arbp and Ppia, is shown on the log₂ scale. For each gene, mean value of two independent experiments with two technical replicates is shown.

FIG. 8 shows quantification of the exogenous TF transcript levels induced by Dox. FIG. 8a , Quantification of the amplification efficiencies of primer pairs used for the amplification of the indicated sequences. FIG. 8b , Expression levels measured by Q-PCR of the exogenous and endogenous Blimp1, Prdm14, and Tfap2c in whole floating aggregates of EpiLCs from the BVSCR26rtTA BP14A cells or the parental BVSCR26rtTA cells induced by Dox, or from the parental BVSCR26rtTA cells induced by the cytokines during the course of 48 hrs. For each gene, the ΔCT from the average CT values of two independent housekeeping genes Arbp and Ppia is shown on the log₂ scale. For each gene, mean value of two independent experiments with two technical replicates is shown. FIG. 8c , Percentage of β-galactosidase-positive cells in dissociated single cells of floating aggregates of EpiLCs from BVSCR26rtTA BP14A, BP14, BA, P14A, B, P14, and A cells with or without Dox for 12 hrs. FIG. 8d, 8e, 8f, 8g , Measurements of the expression levels of β-galactosidase (reflecting whole exogenous TF transcript levels, d), exogenous Blimp1 (e), Prdm14 (f), and Tfap2c (g) in whole floating aggregates of EpiLCs from BVSCR26rtTA BP14A (clones 3-3, 3-6, 3-10), BP14 (clones 4-2, 4-6, 4-7), BA (clones 5-3, 5-4, 5-10), P14A (clones 6-1, 6-2, 6-4, 6-5), B (clones 2-1, 2-4, 2-6), P14 (clones 7-1, 7-5, 7-8), and A (clones 8-2, 8-4, 8-7) cells induced by Dox for 12 hrs. The mean values with standard deviations (SDs) from at least two independent experiments for each line are shown. Note that the whole exogenous transcript levels measured by β-galactosidase are nearly equal to the sum of the levels of each exogenous TF measured by specific primers for each TF expressed in each clone.

FIG. 9 shows FACS of the cells for microarray analysis and for epigenetic profiling, and “core PGC genes” and “somatic mesodermal genes”. FIG. 9a , FACS of the cells used for global transcription analysis by the microarray shown in FIG. 3a . The BV-positive cells delineated by red squares were used for the microarray analysis. FIG. 9b , (top) Genes up-regulated in d2 TF(BP14A)-PGCLCs in comparison to those in EpiLCs/control EpiLCs without exogenous TFs but treated with Dox (“core PGC genes”). (bottom) Genes up-regulated in d2 Ck-PGCLCs but not in d2 TF-PGCLCs in comparison to those in EpiLCs (“somatic mesodermal genes”). The expression levels defined by the microarray analysis are represented by a graded color code as shown at the bottom. The symbols for key genes are shown on the right. At least two independent samples were used for the analysis. FIG. 9c , FACS analysis of EpiLCs and d4 BV-positive TF (BP14A, Line 3-3)-PGCLCs used in FIG. 3 b.

FIG. 10 shows development of embryos and growth of offspring derived from TF (BP14A)-PGCLC-derived spermatozoa. FIG. 10a , A testis (right) transplanted with the SC-positive cells induced by activation of BP14A in ESCs. As a control, an untransplanted testis is shown on the left. Bar, 1 mm. FIG. 10b , A histological section stained by hematoxylin-eosin of a testis bearing the SC-positive cell-derived teratoma. The areas delineated in black squares in the left panel are magnified in the right panels. (right, top) A ciliated endoderm-like cell lining and bone with bone marrow bearing hematopoietic cells are formed. (right, bottom) Well-differentiated neurons and neural fibers are eminent. Bars: (the left panel), 100 μm, (the right panels), 100 μm. FIG. 10c , Development of embryos derived from TF (BP14A)- and Ck-PGCLC-derived spermatozoa. FIG. 10d , Growth of offspring derived from TF (BP14A)- and Ck-PGCLC-derived spermatozoa. FIG. 10e , Fertility of mice (indicated by numbers, FIG. 4i ) derived from TF (BP14A)-derived spermatozoa.

DETAILED DESCRIPTION OF THE INVENTION (I) Method of Producing PGCLCs from Epiblasts or EpiLCs

The present invention provides a method of producing PGCLCs from isolated epiblasts or EpiLCs, which comprises allowing the epiblasts or EpiLCs to express certain exogenously introduced TF(s) that show specific expression in PGCs, without cytokines such as BMP4, LIF, SCF, BMP8b and EGF.

(Ia) Epiblasts and Preparation Thereof

The “epiblast” for use as the starting material is a pre-gastrulating epiblast cell (in mice, from embryonic day (E) 5.5 to E6.0 but not those later than E6.25) derived from inner cell mass of a mammalian (e.g., mouse, human, monkey, rat, rabbit, bovine, horse, porcine, canine, sheep, goat, etc., preferably mouse and human) blastocyst. The epiblasts used in the present invention can be prepared by isolating mammalian embryos in an appropriate buffer, treating the embryos with trypsin/pancreatin, removing visceral endoderm (VE) by pipetting and cutting off extraembryonic ectoderm (ExE) by a glass needle. Isolated epiblasts can be cultured in a serum-free medium supplemented with 10-20% KSR and the like.

(Ib-1) EpiLCs

The “epiblast-like cell (EpiLC)” for use as the starting material is a culture equivalent of pre-gastrulating epiblast cell derived from a pluripotent stem cell (PSC). To be specific, the EpiLC is defined as a cell having ether or both of the following properties:

(1) elevated gene expression of at least one selected from Fgf5, Wnt3 and Dnmt3b compared to the PSC before inducing differentiation;

(2) reduced gene expression of at least one selected from Gata4, Gata6, Sox17 and Blimp1 compared to the PSC before inducing differentiation.

More preferably, the EpiLC of the present invention has the following properties:

(1) continuous gene expression of Oct3/4;

(2) reduced gene expression of Sox2 and Nanog compared to the PSC before inducing differentiation;

(3) elevated gene expression of Fgf5, Wnt3 and Dnmt3b compared to the PSC before inducing differentiation; and

(4) reduced gene expression of Gata4, Gata6, Sox17 and Blimp1 compared to the PSC before inducing differentiation.

(Ib-2) Induction of Differentiation from PSCs to EpiLCs

The EpiLC of the present invention can be prepared from a PSC as previously described (WO 2012/020687; Cell 146, 519-532 (2011); Science 338, 971-975 (2012)).

(Ib-2(i)) PSCs and Preparation Thereof

The “pluripotent stem cell (PSC)” for use as the starting, material may be any undifferentiated cell possessing a “self-renewal” that enables it to proliferate while retaining the undifferentiated state, and “pluripotency” that enables it to differentiate into all the three primary germ layers of the embryo. Examples include embryonic stem (ES) cell, embryonic stem cell derived from a cloned embryo obtained by nuclear transplantation (ntES cell), germline stem cell (“GS cell”), embryonic germ cell (“EG cell”), induced pluripotent stem (iPS) cell, cultured fibroblast- or myeloid stem cell-derived pluripotent cell (Muse cell) and the like. Preferable pluripotent stem cells are ES cell, ntES cell and iPS cell.

(A) Embryonic Stem Cell

ES cell is a stem cell having pluripotency and proliferation potency based on self-renewal, which is established from an inner cell mass of an early-stage embryo (for example, blastocyst) of a mammal such as human, mouse and the like.

ES cell is an embryo-derived stem cell derived from an inner cell mass of blastocyst, which is an embryo after morula at 8-cell stage of a fertilized egg, and has an ability to differentiate into any cell constituting an adult body, i.e., pluripotent differentiation potency, and proliferation potency based on self-renewal. The ES cell was discovered in mouse in 1981 (M. J. Evans and M. H. Kaufman (1981), Nature 292:154-156) and thereafter ES cell lines were also established in primates such as human, monkey and the like (J. A. Thomson et al. (1998), Science 282:1145-1147; J. A. Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848; J. A. Thomson et al. (1996), Biol. Reprod., 55: 254-259; J. A. Thomson and V. S. Marshall (1998), Curt. Top. Dev. Biol., 38:133-165).

ES cell can be established by removing an inner cell mass from the blastocyst of a fertilized egg of a target animal, and culturing the inner cell mass on fibroblast feeder cells. In addition, the cells can be maintained by passage culture using a culture medium added with substances such as leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and the like. The methods of establishment and maintenance of human and monkey ES cells are described in, for example, U.S. Pat. No. 5,843,780; Thomson J A, et al. (1995), Proc Natl. Acad. Sci. USA. 92:7844-7848; Thomson J A, et al. (1998), Science. 282:1145-1147; H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345:926-932; M. Ueno et al. (2006), Proc. Natl. Acad. Sci. USA, 103:9554-9559; H. Suemori et al. (2001), Dev. Dyn., 222:273-279; H. Kawasaki et al. (2002), Proc. Natl. Acad. Sci. USA, 99:1580-1585; Klimanskaya I, et al. (2006), Nature. 444:481-485 and the like.

Using, as a culture medium for preparing ES cells, for example, a DMEM/F-12 culture medium supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM nonessential amino acids, 2 mM L-glutamic acid, 20% KSR and 4 ng/ml bFGF, human ES cells can be maintained under wet atmosphere at 37° C., 2% CO₂/98% air (O. Fumitaka et al. (2008), Nat. Biotechnol., 26:215-224). In addition, ES cells require passage every 3-4 days, and the passage in this case can be performed using, for example, 0.25% trypsin and 0.1 mg/ml collagenase IV in PBS containing 1 mM CaCl₂ and 20% KSR.

ES cells can be generally selected by the Real-Time PCR method using the expression of a gene marker such as alkaline phosphatase, Oct-3/4, Nanog and the like as an index. Particularly, for selection of human ES cell, expression of a gene marker such as OCT-3/4, NANOG, ECAD and the like can be used as an index (E. Kroon et al. (2008), Nat. Biotechnol., 26:443-452).

As for human ES cell line, for example, WA01(H1) and WA09(H9) are available from WiCell Research Institute, and KhES-1, KhES-2 and KhES-3 are available from Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan).

(B) Germline Stem Cell

Germline stem cell is a pluripotent stem cell derived from the testis, which becomes the origin for spermatogenesis. This cell can be differentiation induced into various lines of cells, like ES cells and shows properties of, for example, generation of a chimeric mouse by transplantation into a mouse blastocyst and the like (M. Kanatsu-Shinohara et al. (2003) Biol. Reprod., 69:612-616; K. Shinohara et al. (2004), Cell, 119:1001-1012). It is self-renewable in a culture medium containing a glial cell line-derived neurotrophic factor (GDNF), can produce a germline stem cell by repeating passages under culture conditions similar to those for ES cells (Masanori Takehashi et al., (2008), Experimental Medicine, Vol. 26, No. 5(Suppl.), pp. 41-46, YODOSHA (Tokyo, Japan)).

(C) Embryonic Germ Cell

Embryonic germ cell is a cell having pluripotency similar to that of ES cells, which is established from a primordial germ cell at the prenatal period. It can be established by culturing a primordial germ cell in the presence of a substance such as LIF, bFGF, a stem cell factor and the like (Y. Matsui et al. (1992), Cell, 70:841-847; J. L. Resnick et al. (1992), Nature, 359:550-551).

(D) Induced Pluripotent Stem Cell

Induced pluripotent stem (iPS) cell is an artificial stem cell derived from a somatic cell, which can be produced by introducing a specific reprogramming factor in the form of a DNA or protein into a somatic cell, and show almost equivalent property (e.g., pluripotent differentiation and proliferation potency based on self-renewal) as ES cells (K. Takahashi and S. Yamanaka (2006) Cell, 126:663-676; K. Takahashi et al. (2007), Cell, 131:861-872; J. Yu et al. (2007), Science, 318:1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26:101-106 (2008); WO2007/069666). The reprogramming factor may be constituted with a gene specifically expressed by ES cell, a gene product or non-coding RNA thereof, a gene playing an important role for the maintenance of undifferentiation of ES cell, a gene product or non-coding RNA thereof, or a low molecular weight compound. Examples of the gene contained in the reprogramming factor include Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sal11, Sal14, Esrrb, Nr5a2, TbX3, Glis1 and the like. These reprogramming factors may be used alone or in combination. Examples of the combination of the reprogramming factors include combinations described in WO2007/069666, WO2008/118820, WO2009/007852, WO2009/032194, WO2009/058413, WO2009/057831, WO2009/075119, WO2009/079007, WO2009/091659, WO2009/101084, WO2009/101407, WO2009/102983, WO2009/114949, WO2009/117439, WO2009/126250, WO2009/126251, WO2009/126655, WO2009/157593, WO2010/009015, WO2010/033906, WO2010/033920, WO2010/042800, WO2010/050626, WO2010/056831, WO2010/068955, WO2010/098419, WO2010/102267, WO2010/111409, WO2010/111422, WO2010/115050, WO2010/124290, WO2010/147395, WO2010/147612, Huangfu D, et al. (2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26:2467-2474, Huangfu D, et al. (2008), Nat Biotechnol. 26:1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat Cell Biol. 11:197-203, R. L. Judson et al., (2009), Nat. Biotech., 27:459-461, Lyssiotis C A, et al. (2009), Proc Natl Acad Sci USA. 106:8912-8917, Kim J B, et al. (2009), Nature. 461:649-643, Ichida J K, et al. (2009), Cell Stem Cell. 5:491-503, Heng J C, et al. (2010), Cell Stem Cell. 6:167-74, Han J, et al. (2010), Nature. 463:1096-100, Mali P, et al. (2010), Stem Cells. 28:713-720, and Maekawa M, et al. (2011), Nature. 474:225-9.

Examples of the above-mentioned reprogramming factor include, but are not limited to, factors used for enhancing the establishment efficiency, such as histone deacetylase (HDAC) inhibitors [e.g., low-molecular inhibitors such as valproic acid (VPA), trichostatin A, sodium butyrate, MC 1293, and M344, nucleic acid-based expression inhibitors such as siRNAs and shRNAs against HDAC (e.g., HDAC1 siRNA Smartpool® (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene) and the like), and the like], MEK inhibitor (e.g., PD184352, PD98059, U0126, SL327 and PD0325901), Glycogen synthase kinase-3 inhibitor (e.g., Bio and CHIR99021), DNA methyl transferase inhibitors (e.g., 5-azacytidine), histone methyl transferase inhibitors [for example, low-molecular inhibitors such as BIX-01294, and nucleic acid-based expression inhibitors such as siRNAs and shRNAs against Suv39h1, Suv39h2, SetDB1 and G9a], L-channel calcium agonist (for example, Bayk8644), butyric acid, TGFβ inhibitor or ALK5 inhibitor (e.g., LY364947, SB431542, 616453 and A-83-01), p53 inhibitor (for example, siRNA and shRNA against p53), ARID3A inhibitor (e.g., siRNA and shRNA against ARID3A), miRNA such as miR-291-3p, miR-294, miR-295, mir-302 and the like, Wnt Signaling (for example, soluble Wnt3a), neuropeptide Y, prostaglandins (e.g., prostaglandin E2 and prostaglandin J2), hTERT, SV40LT, UTF1, IRX6, GLIS1, PITX2, DMRTB1 and the like. In the present specification, these factors used for enhancing the establishment efficiency are not particularly distinguished from the reprogramming factor.

When the reprogramming factor is in the form of a protein, it may be introduced into a somatic cell by a method, for example, lipofection, fusion with cell penetrating peptide (e.g., TAT derived from HIV and polyarginine), microinjection and the like.

When the reprogramming factor is in the form of a DNA, it may be introduced into a somatic cell by the method using, for example, vector of virus, plasmid, artificial chromosome and the like, lipofection, liposome, microinjection and the like. Examples of the virus vector include retrovirus vector, lentivirus vector (Cell, 126, pp. 663-676, 2006; Cell, 131, pp. 861-872, 2007; Science, 318, pp. 1917-1920, 2007), adenovirus vector (Science, 322, 945-949, 2008), adeno-associated virus vector, Sendai virus vector (vector of Hemagglutinating Virus of Japan) (WO 2010/008054) and the like. Examples of the artificial chromosome vector include human artificial chromosome (HAC), yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC, PAC) and the like. As the plasmid, plasmids for mammalian cells can be used (Science, 322:949-953, 2008). The vector can contain regulatory sequences of promoter, enhancer, ribosome binding sequence, terminator, polyadenylation site and the like so that a nuclear reprogramming substance can be expressed and further, where necessary, a selection marker sequence of a drug resistance gene (for example, kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene and the like), thymidine kinase gene, diphtheria toxin gene and the like, a reporter gene sequence of green fluorescent protein (GFP), β glucuronidase (GUS), FLAG and the like, and the like. Moreover, the above-mentioned vector may have a LoxP sequence before and after thereof to simultaneously cut out a gene encoding a reprogramming factor or a gene encoding a reprogramming factor bound to the promoter, after introduction into a somatic cell.

When in the form of RNA, for example, it may be introduced into a somatic cell by means of lipofection, microinjection and the like, and RNA incorporating 5-methylcytidine and pseudouridine (TriLink Biotechnologies) may be used to suppress degradation (Warren L, (2010) Cell Stem Cell. 7:618-630).

Examples of the culture medium for inducing iPS cell include 10-15% FBS-containing DMEM, DMEM/F12 or DME culture medium (these culture media can further contain LIF, penicillin/streptomycin, puromycin, L-glutamine, nonessential amino acids, β-mercaptoethanol and the like as appropriate) or a commercially available culture medium [for example, culture medium for mouse ES cell culture (TX-WES culture medium, Thromb-X), culture medium for primate ES cell (culture medium for primate ES/iPS cell, Reprocell), serum-free medium (mTeSR, Stemcell Technologies)] and the like.

Examples of the culture method include contacting a somatic cell with a reprogramming factor on 10% FBS-containing DMEM or DMEM/F12 culture medium at 37° C. in the presence of 5% CO₂ and culturing for about 2-7 days, thereafter reseeding the cells on feeder cells (e.g., mitomycin C-treated STO cells, SNL cells etc.), and culturing the cells in a bFGF-containing culture medium for primate ES cell from about 10 days after the contact of the somatic cell and the reprogramming factor, whereby iPS-like colonies can be obtained after about 30-about 45 days or longer from the contact.

Alternatively, the cells are cultured on feeder cells (e.g., mitomycin C-treated STO cells, SNL cells etc.) at 37° C. in the presence of 5% 002 in a 10% FBS-containing DMEM culture medium (which can further contain LIF, penicillin/streptomycin, puromycin, L-glutamine, nonessential amino acids, β-mercaptoethanol and the like as appropriate), whereby ES-like colonies can be obtained after about 25-about 30 days or longer. Desirably, a method using a somatic cell itself to be reprogrammed, or an extracellular substrate (e.g., Laminin-5 (WO2009/123349) and Matrigel (BD)), instead of the feeder cells (Takahashi K, et al. (2009), PLoS One. 4:e8067 or WO2010/137746), can be mentioned.

Besides the above, a culture method using a serum-free medium can also be recited as an example (Sun N, et al. (2009), Proc Natl Acad Sci USA. 106:15720-15725). Furthermore, to enhance establishment efficiency, an iPS cell may be established under hypoxic conditions (oxygen concentration of not less than 0.1% and not more than 15%) (Yoshida Y, et al. (2009), Cell Stem Cell. 5:237-241 or WO2010/013845).

The culture medium is exchanged with a fresh culture medium once a day during the above-mentioned cultures, from day 2 from the start of the culture. While the cell number of the somatic cells used for nuclear reprogramming is not limited, it is about 5×10³-about 5×10⁶ cells per 100 cm² culture dish.

The iPS cell can be selected based on the shape of the formed colony. When a drug resistance gene which is expressed in association with a gene (e.g., Oct3/4, Nanog) expressed when a somatic cell is reprogrammed is introduced as a marker gene, an established iPS cell can be selected by culturing in a culture medium (selection culture medium) containing a corresponding drug. When the marker gene is a fluorescent protein gene, iPS cell can be selected by observation with a fluorescence microscope, when it is a luminescent enzyme gene, iPS cell can be selected by adding a luminescent substrate, and when it is a chromogenic enzyme gene, iPS cell can be selected by adding a chromogenic substrate.

The term “somatic cell” used in the present specification means any animal cell (preferably, cells of mammals inclusive of human) excluding germ line cells and totipotent cells such as ovum, oocyte, ES cells and the like. Somatic cell unlimitatively encompasses any of somatic cells of fetuses, somatic cells of neonates, and mature healthy or pathogenic somatic cells, and any of primary cultured cells, passage cells, and established lines of cells. Specific examples of the somatic cell include (1) tissue stem cells (somatic stem cells) such as neural stem cell, hematopoietic stem cell, mesenchymal stem cell, dental pulp stem cell and the like, (2) tissue progenitor cell, (3) differentiated cells such as lymphocyte, epithelial cell, endothelial cell, myocyte, fibroblast (skin cells etc.), hair cell, hepatocyte, gastric mucosal cell, enterocyte, splenocyte, pancreatic cell (pancreatic exocrine cell etc.), brain cell, lung cell, renal cell and adipocyte and the like, and the like.

The choice of mammal individual as a source of somatic cells is not particularly limited; however, when the PGC-like cells as a final product are to be used for the treatment of diseases such as infertility in humans, it is preferable, from the viewpoint of prevention of graft rejection and/or GvHD, that somatic cells are patient's own cells or collected from another person having the same or substantially the same HLA type as that of the patient. “Substantially the same HLA type” as used herein means that the HLA type of donor matches with that of patient to the extent that the transplanted cells, which have been obtained by inducing differentiation of iPS cells derived from the donor's somatic cells, can be engrafted when they are transplanted to the patient with use of immunosuppressor and the like. For example, it includes an HLA type wherein major HLAs (the three major loci of HLA-A, HLA-B and HLA-DR or four loci further including HLA-Cw) are identical (hereinafter the same meaning shall apply) and the like. When the PGC-like cells are not to be administered (transplanted) to a human, but used as, for example, a source of cells for screening for evaluating a patient's drug susceptibility or adverse reactions, it is likewise necessary to collect the somatic cells from the patient or another person with the same genetic polymorphism correlating with the drug susceptibility or adverse reactions.

(E) Naive Human ES and iPS Cells

Conventional human ES cells derived from blastocyst-stage embryos have very different biological (morphological, molecular and functional) properties from mouse ES cells. Mouse pluripotent stem cells can exist in two functionally distinct states, LIF-dependent ES cells and bFGF-dependent epiblast stem cells (EpiSCs). Molecular analyses suggest that the pluripotent state of human ES cells is similar to that of mouse EpiSCs rather than that of mouse ES cells. Recently, human ES and iPS cells in a mouse ES cell-like pluripotent state (also refereed to as naive human ES and iPS cells) have been established by ectopic induction of Oct3/4, Sox2, Klf4, c-Myc and Nanog in the presence of LIF (see Cell Stem Cells, 6: 535-546, 2010), or ectopic induction of Oct3/4, Klf4 and Klf2 combined with LIF and inhibitors of GSK3β and ERK1/2 pathway (see Proc. Natl. Acad. Sci. USA, online publication doi/10.1073/pnas.1004584107). These naive human ES and iPS cells may be preferable starting materials for the present invention due to their pluripotent more immature compared to that of conventional human ES and iPS cells.

(F) ES Cells Derived from Cloned Embryo by Nuclear Transplantation

nt ES cell is an ES cell derived from a cloned embryo prepared by a nuclear transplantation technique, and has almost the same property as the ES cell derived from a fertilized egg (T. Wakayama et al. (2001), Science, 292:740-743; S. Wakayama et al. (2005), Biol. Reprod., 72:932-936; J. Byrne et al. (2007), Nature, 450:497-502). That is, an ES cell established from an inner cell mass of a blastocyst derived from a cloned embryo obtained by substituting the nucleus of an unfertilized egg with the nucleus of a somatic cell is an nt ES (nuclear transfer ES) cell. For production of an nt ES cell, a combination of the nuclear transplantation technique (J. B. Cibelli et al. (1998), Nature Biotechnol., 16:642-646) and the ES cell production technique (mentioned above) is used (Kiyoka Wakayama et al., (2008), Experimental Medicine, Vol. 26, No. 5(Suppl.), pp. 47-52). In nuclear transplantation, reprogramming can be performed by injecting the nucleus of a somatic cell to an enucleated unfertilized egg of a mammal, and culturing for a few hours.

(G) Multilineage-Differentiating Stress Enduring Cell (Muse Cell)

Muse cell is a pluripotent stem cell produced by the method described in WO2011/007900. In more detail, it is a cell having pluripotency, which is obtained by subjecting a fibroblast or a bone marrow stromal cell to a trypsin treatment for a long time, preferably 8 hr or 16 hr, and thereafter culturing the cells in a suspended state, and positive for SSEA-3 and CD105.

(Ib-2(ii)) Induction of Differentiation from PSCs to EpiLCs

Basal media for differentiation induction include, but are not limited to, Neurobasal medium, Neural Progenitor Basal medium, NS-A medium, BME medium, BGJb medium, CMRL 1066 medium, minimal essential medium (MEM), Eagle MEM, αMEM, Dulbecco's modified Eagle medium (DMEM), Glasgow MEM, Improved MEM Zinc Option medium, IMDM medium, 199 medium, DMEM/F12 medium, Ham's medium, RPMI1640 medium, Fischer's medium, and mixtures thereof.

The medium can be a serum-containing or serum-free medium. Preferably, a serum-free medium can be used. The serum-free medium (SFM) refers to media with no unprocessed or unpurified serum and accordingly, can include media with purified blood-derived components or animal tissue-derived components (such as growth factors). The concentration of serum (for example, fetal bovine serum (FBS), human serum, etc.) can be 0-20%, preferably 0-5%, more preferably 0-2%, most preferably 0% (i.e., serum-free). The SFM may contain or may not contain any alternatives to serum. The alternatives to serum can include materials which appropriately contain albumin (such as lipid-rich albumin, albumin substitutes such as recombinant albumin, plant starch, dextrans and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3′-thiolglycerol, or equivalents thereto. The alternatives to serum can be prepared by the method disclosed in WO 98/30679, for example. Alternatively, any commercially available materials can be used for more convenience. The commercially available materials include Knockout™ Serum Replacement (KSR), Chemically-defined Lipid concentrated, and Glutamax (Invitrogen).

The medium can also contain other additives known per se. The additive is not subject to limitation, as long as EpiLCs equivalent to pre-gastrulating epiblast cells can be produced by the method of the present invention; for example, growth factors (for example, insulin and the like), polyamines (for example, putrescine and the like), minerals (for example, sodium selenate and the like), saccharides (for example, glucose and the like), organic acids (for example, pyruvic acid, lactic acid and the like), amino acids (for example, non-essential amino acids (NEAA), L-glutamine and the like), reducing agents (for example, 2-mercaptoethanol and the like), vitamins (for example, ascorbic acid, d-biotin and the like), steroids (for example, [beta]-estradiol, progesterone and the like), antibiotics (for example, streptomycin, penicillin, gentamycin and the like), buffering agents (for example, HEPES and the like), nutritive additives (for example, B27 supplement, N2 supplement, StemPro-Nutrient Supplement and the like) and the like can be mentioned. It is preferable that each of the additives be contained in a concentration range known per se.

In the method of producing EpiLCs of the present invention, pluripotent stem cells may be cultured in the presence or absence of feeder cells. The feeder cells are not subject to limitation, as long as EpiLCs can be produced by the method of the present invention; feeder cells known per se for use in culturing pluripotent stem cells such as ESCs and iPSCs can be used; for example, fibroblasts (mouse embryonic fibroblasts, mouse fibroblast cell line STO and the like) can be mentioned. The feeder cells are preferably inactivated by a method known per se, for example, radiation (gamma rays and the like), treatment with an anticancer agent (mitomycin C and the like) and the like. However, in a preferable embodiment of the present invention, pluripotent stem cells are cultured under feeder-free conditions.

The medium for inducing differentiation from pluripotent stem cells to EpiLCs (medium A) contains activin A as an essential additive in the basal medium. The activin A concentration is, for example, about 5 ng/ml or more, preferably about 10 ng/ml or more, more preferably about 15 ng/ml or more, and is, for example, about 40 ng/ml or less, preferably about 30 ng/ml or less, more preferably 25 ng/ml or less.

The medium A preferably further contains bFGF and/or KSR. Basic FGF and KSR remarkably increase the induction efficiency for EpiLCs when present in a range of effective concentrations. The bFGF concentration is, for example, about 5 ng/ml or more, preferably about 7.5 ng/ml or more, more preferably about 10 ng/ml or more, and is, for example, about 30 ng/ml or less, preferably about 20 ng/ml or less, more preferably about 15 ng/ml or less. The KSR concentration is, for example, about 0.1 w/w % or more, preferably about 0.3 w/w % or more, more preferably about 0.5 w/w % or more, and is, for example, about 5 w/w % or less, preferably about 3 w/w % or less, more preferably about 2 w/w % or less.

In a particularly preferred embodiment, the medium A contains activin A, bFGF and KSR in addition to the basal medium. Appropriate concentrations of these ingredients can be chosen over the range of about 10-30 ng/ml, preferably 15-25 ng/ml for activin A, about 7.5-20 ng/ml, preferably about 10-15 ng/ml for bFGF, and about 0.3-3 w/w %, preferably about 0.5-2 w/w % for KSR.

The activin A and bFGF contained in the medium A are not subject to limitation as to the source thereof, may be isolated and purified from cells of any mammals (for example, human, mouse, monkey, swine, rat, dog and the like). It is preferable to use activin A and bFGF homologous to the pluripotent stem cells subjected to the culture. The activin A and bFGF may also be chemically synthesized or biochemically synthesized using a cell-free translation system, or produced from a transformant bearing a nucleic acid encoding each of the proteins. The recombinant products of activin A and bFGF are commercially available.

A culture vessel used for inducing pluripotent stem cells into EpiLCs can include, but is particularly not limited to, flask, flask for tissue culture, dish, petri dish, dish for tissue culture, multi dish, micro plate, micro-well plate, multi plate, multi-well plate, micro slide, chamber slide, schale, tube, tray, culture bag, and roller bottle. The culture vessel can be cellular adhesive. The cellular adhesive culture vessel can be coated with any of substrates for cell adhesion such as extracellular matrix (ECM) to improve the adhesiveness of the vessel surface to the cells. The substrate for cell adhesion can be any material intended to attach pluripotent stem cells or feeder cells (if used). The substrate for cell adhesion includes collagen, gelatin, poly-L-lysine, poly-D-lysine, poly-L-orthinine, laminin, and fibronectin and mixtures thereof for example Matrigel, and lysed cell membrane preparations (Klimanskaya I et al 2005. Lancet 365: p 1636-1641).

In this cultivation, pluripotent stem cells are plated onto the culture vessel mentioned above to obtain a cell density of, for example, about 10⁴-10⁵ cells/cm², preferably about 2 to 8×10⁴ cells/cm², and cultured in an incubator under atmospheric conditions of 1-10% CO₂/99-90% air at about 30-40° C., preferably about 37° C., for less than 3 days, preferably about 2 days (e.g., 48±12 hours, preferably 48±6 hours). As a result of the culture, cells with flattened epiblast-like structure uniformly emerge.

The fact of differentiation into EpiLCs can be confirmed by, for example, analyzing the expression levels of EPiLC- and/or pluripotent stem cell-marker genes using RT-PCR. As mentioned above, EpiLC is defined as a cell with (1) elevated gene expression of at least one selected from Fgf5, Wnt3 and Dnmt3b compared to the PSC before inducing differentiation and (2) reduced gene expression of at least one selected from Gata4, Gata6, Sox17 and Blimp1 compared to the PSC before inducing differentiation. Therefore, the fact of differentiation into EpiLCs can be confirmed by determining the expression levels of at least one selected from Fgf5, Wnt3 and Dnmt3b and/or at least one selected from Gata4, Gata6, Sox17 and Blimp1 in the cells obtained by the culture, and comparing the expression levels with those in the pluripotent stem cells before inducing differentiation.

(Ic) Induction of Differentiation from Epiblasts or EpiLCs to PGCLCs

By allowing thus-obtained epiblasts or EpiLCs to express certain exogenously introduced TF(s) that show specific expression in PGCs, it is possible to induce the epiblasts or EpiLCs into PGC-like cells (PGCLCs) without cytokines including BMP4.

The combinations of TF(s) capable of inducing the epiblasts or EpiLCs into PGLCs include the following (i) to (v):

(i) Blimp1, Prdm14 and Tfap2c;

(ii) Blimp1 and Prdm14;

(iii) Blimp1 and Tfap2c;

(iv) Prdm14 and Tfap2c; and

(v) Prdm14.

Hereinafter these TFs are also referred to as “the PGCLC inducer of the present invention”. The PGCLC inducer of the present invention may be introduced in a form of protein or nucleic acid encoding same into an epiblast or EpiLC.

As the PGCLC inducer of the present invention, for example, a Blimp1, Prdm14 or Tfap2c protein derived from any mammal (e.g., human, mouse, rat, monkey, bovine, horse, swine, dog etc.) or a nucleic acid encoding the same and the like can be used. A species of the same derivation as the target epiblast or EpiLC is preferable.

Examples of the Blimp1 to be used in the present invention include human B-lymphocyte-induced maturation protein 1 (BLIMP1) consisting of the amino acid sequence shown by SEQ ID NO: 2 (registered as NCBI accession number: NP_001189.2), mouse Blimp1 consisting of the amino acid sequence shown by SEQ ID NO: 4 (registered as NCBI accession number: NP_031574.1), and an ortholog thereof in other mammal (see GeneCards® human gene database), a polymorphic variant thereof (e.g., G74S (dbSNP No.: rs2185379), D203E (dbSNP No.: rs811925), a splicing variant thereof (e.g., 075626-2, 075626-3; see UniProtKB/Swiss-Prot database) and the like. Alternatively, it may be a protein having an amino acid identity of not less than 90%, preferably not less than 95%, more preferably not less than 97%, even more preferably not less than 98%, most preferably not less than 99%, with any of the above-mentioned proteins, and having a function equivalent to that of said protein (e.g., transcription activation of PGC-specific promoter and the like). The identity of the amino acid sequence as mentioned herein can be calculated using the blastp program of the NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expectancy=10; gap allowed; matrix=BLOSUM62; filtering=OFF).

Examples of Prdm14 to be used in the present invention include human PR domain containing 14 (PRDM14) consisting of the amino acid sequence shown by SEQ ID NO: 6 (registered as NCBI accession number: NP_078780.1), mouse Prdm14 consisting of the amino acid sequence shown by SEQ ID NO: 8 (registered as NCBI accession number: NP_001074678.1), and an ortholog thereof in other mammal (see GeneCards® human gene database), a polymorphic variant thereof, a splicing variant thereof and the like. Alternatively, it may be a protein having an amino acid identity m of not less than 90%, preferably not less than 95%, more preferably not less than 97%, even more preferably not less than 98%, most preferably not less than 99%, with any of the above-mentioned proteins, and having a function equivalent to that of said protein (e.g., transcription activation of PGC-specific promoter and the like). Here, the identity of the amino acid sequence can be calculated in the same manner as in the above.

Examples of Tfap2c to be used in the present invention include human transcription factor AP-2 gamma (TFAP2C) consisting of the amino acid sequence shown by SEQ ID NO: 10 (registered as NCBI accession number: NP_003213.1), mouse Tfap2c consisting of the amino acid sequence shown by SEQ ID NO: 12 (registered as NCBI accession number: NP_033361.1), and an ortholog thereof in other mammal (see GeneCards® human gene database), a polymorphic variant thereof (e.g., K244E; see Genome Res. 14:2121-2127(2004)), a splicing variant thereof and the like. Alternatively, it may be a protein having an amino acid identity of not less than 90%, preferably not less than 95%, more preferably not less than 97%, even more preferably not less than 98%, most preferably not less than 99%, with any of the above-mentioned proteins, and having a function equivalent to that of said protein (e.g., transcription activation of PGC-specific promoter and the like). Here, the identity of the amino acid sequence can be calculated in the same manner as in the above.

Blimp1, Prdm14 or Tfap2c may be a fusion protein of any of the above-mentioned proteins and a cell penetrating peptide (e.g., TAT derived from HIV and polyarginine).

Examples of the nucleic acid encoding Blimp1 include human B-lymphocyte-induced maturation protein 1 (BLIMP1) cDNA consisting of the nucleotide sequence shown by SEQ ID NO: 1 (registered as NCBI accession number: NM_001198.3), mouse Blimp1 cDNA consisting of the nucleotide sequence shown by SEQ ID NO: 3 (registered as NCBI accession number: NM_007548.2), and an ortholog thereof in other mammal, a transcription variant thereof, a splicing variant thereof and the like. Alternatively, it may be a nucleic acid encoding a protein having a nucleotide identity of not less than 90%, preferably not less than 95%, more preferably not less than 97%, even more preferably not less than 98%, most preferably not less than 99%, with any of the above-mentioned nucleic acids, and having a function equivalent to that of a protein encoded by said nucleic acid (e.g., transcription activation of PGC-specific promoter and the like). The identity of the nucleotide sequence as mentioned herein can be calculated using the blastn program of the NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expectancy=10; gap allowed; filtering-ON; match score=1; mismatch score=−3). Alternatively, it may have a plus strand in a complementary relationship of the level permitting hybridization with the complementary strand of any of the above-mentioned nucleic acids under stringent conditions. The stringent conditions herein can be determined based on the melting temperature (Tm) of the nucleic acid binding to a complex or probe, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego Calif.). For example, wash conditions after hybridization generally include about “1×SSC, 0.1% SDS, 37° C.”. The complementary strand is preferably one that maintains hybridization state with the target plus strand even when washed under such conditions. Although not particularly limited, more stringent hybridization conditions include wash conditions of about “0.5×SSC, 0.1% SDS, 42° C.”, more stringent wash conditions of about “0.1×SSC, 0.1% SDS, 65° C.”, which permit the plus strand and the complementary strand to maintain hybridization state even after washing.

Examples of the nucleic acid encoding Prdm14 include human PR domain containing 14 (PRDM14) cDNA consisting of the nucleotide sequence shown by SEQ ID NO: 5 (registered as NCBI accession number: NM_024504), mouse Prdm14 cDNA consisting of the nucleotide sequence shown by SEQ ID NO: 7 (registered as NCBI accession number: NM_001081209), and an ortholog thereof in other mammal, a transcription variant thereof, a splicing variant thereof and the like. Alternatively, it may be a nucleic acid encoding a protein having a nucleotide identity of not less than 90%, preferably not less than 95%, more preferably not less than 97%, even more preferably not less than 98%, most preferably not less than 99%, with any of the above-mentioned nucleic acids, and having a function equivalent to that of a protein encoded by said nucleic acid (e.g., transcription activation of PGC-specific promoter and the like). The identity of the nucleotide sequence as mentioned herein can be calculated in the same manner as mentioned above. Alternatively, it may have a plus strand in a complementary relationship of the level permitting hybridization with the complementary strand of any of the above-mentioned nucleic acids under stringent conditions. The stringent conditions as mentioned herein are as defined above.

Examples of the nucleic acid encoding Tfap2C include human transcription factor AP-2 gamma (TFAP2C) cDNA consisting of the nucleotide sequence shown by SEQ ID NO: 9 (registered as NCBI accession number: NM_003222), mouse Tfap2C cDNA consisting of the nucleotide sequence shown by SEQ ID NO: 11 (registered as NCBI accession number: NM_009335), and an ortholog thereof in other mammal, a transcription variant thereof, a splicing variant thereof and the like. Alternatively, it may be a nucleic acid encoding a protein having a nucleotide identity of not less than 90%, preferably not less than 95%, more preferably not less than 97%, even more preferably not less than 98%, most preferably not less than 99%, with any of the above-mentioned nucleic acids, and having a function equivalent to that of a protein encoded by said nucleic acid (e.g., transcription activation of PGC-specific promoter and the like). The identity of the nucleotide sequence as mentioned herein can be calculated in the same manner as mentioned above. Alternatively, it may have a plus strand in a complementary relationship of the level permitting hybridization with the complementary strand of any of the above-mentioned nucleic acids under stringent conditions. The stringent conditions as mentioned herein are as defined above.

The nucleic acid encoding Blimp1, Prdm14 or Tfap2c may be DNA, RNA or DNA/RNA chimera. In addition, the nucleic acid may be a single strand, double stranded DNA, double stranded RNA or DNA:RNA hybrid. Preferred is a double stranded DNA or single stranded RNA. As said RNA, RNA incorporating 5-methylcytidine and pseudouridine (TriLink Biotechnologies), or a modified RNA obtained by a phosphatase treatment may be used for the suppression of degradation.

Blimp1, Prdm14 and Tfap2c, and nucleic acids encoding them can be obtained by easily isolating a nucleic acid encoding each protein or, where necessary, producing a recombinant protein or chemically synthesizing the same based on, for example, the cDNA sequence information of the above-mentioned human or mouse Blimp1, Prdm14 and Tfap2c.

While the method of expressing a PGCLC inducer of the present invention in an epiblast or EpiLC is not particularly limited, for example, the following method can be used. Here, “expression” means that, when the PGCLC inducer is nucleic acid encoding Blimp1, Prdm14 or Tfap2c, a Blimp1, Prdm14 or Tfap2c protein is produced by intracellular (transcription and) translation from the nucleic acid, and when the PGCLC inducer is a Blimp1, Prdm14 or Tfap2c protein, it means the same as an intracellular introduction of the protein.

When the aforementioned PGCLC inducer is in the form of a DNA, for example, a vector such as virus, plasmid, artificial chromosome and the like may be introduced into an epiblast or EpiLC by a method such as lipofection, liposome, microinjection and the like. Examples of the viral vector include retroviral vector, lentiviral vector, adenoviral vector, adeno-associated viral vector, Sendai viral vector and the like. Examples of the artificial chromosome vector include human artificial chromosome (HAC), yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC, PAC) and the like. Examples of the plasmid include plasmids for mammalian cells. The vector can contain regulatory sequences of promoter, enhancer, ribosome binding sequence, terminator, polyadenylation site and the like so that a DNA encoding Blimp1, Prdm14 or Tfap2c can be expressed and further, where necessary, selectable marker sequences such as a drug resistance gene (for example, kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene and the like), thymidine kinase gene, diphtheria toxin gene and the like, a reporter gene sequence such as fluorescent protein, β-glucuronidase (GUS), FLAG and the like, and the like. As a promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney mouse leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, EF-α promoter, CAG promoter and TRE promoter (minimal CMV promoter having a Tet response element with continuous 7 tetO sequences). When a TRE promoter is used, a fusion protein of tetR and VP16AD or a fusion protein of reverse tetR (rtetR) and VP16AD is desirably expressed simultaneously in the same cell. Here, a vector having a TRE promoter and capable of expressing a fusion protein of reverse tetR (rtetR) and VP16AD is referred to as a drug responsive inducible vector. In addition, to introduce an expression cassette comprising a promoter and a DNA encoding Blimp1, Prdm14 or Tfap2c operably linked thereto into a chromosome of an epiblast or EpiLC and cut it out as necessary therefrom, the above-mentioned vector may have a transposon sequence before and after the expression cassette. While the transposon sequence is not particularly limited, piggyBac can be mentioned. In another embodiment, it may have a LoxP sequence before and after the expression cassette to remove the expression cassette.

When the aforementioned PGCLC inducer is in the form of an RNA, it may be introduced into an epiblast or EpiLC by a method such as electroporation, lipofection, microinjection and the like. When the PGCLC inducer is in the form of a protein, it may be introduced into an epiblast or EpiLC by a method such as lipofection, fusion with cell penetrating peptide (e.g., TAT derived from HIV and polyarginine), microinjection and the like.

An exogenous PGCLC inducer may be expressed in an epiblast or EpiLC at least within 3 days from the start of the culture of the epiblast or EpiLC for PGCLC induction (i.e., culture in the absence of cytokines including BMP4), desirably within 1 day from the start of the culture, more desirably immediately after the start of the culture. When a PGCLC inducer is expressed after 3 days from the start of the culture for PGCLC induction, the PGCLC induction efficiency may decrease. While the period when the expression of an exogenous PGCLC inducer is maintained is not particularly limited, it is desirably not less than 1 day and preferably not more than 10 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days), more preferably 1 to 6 days, even more preferably 1 to 5 days, most preferably 2 to 4 days (e.g., 2, 3 or 4 days). While the method of maintaining the expression is not particularly limited, when the PGCLC inducer is a non-integrated and non-episomally replicable molecule such as plasmid, RNA or protein, the introduction can be performed plural times during a desired period (for example, once every 2 days for plasmid introduction, once everyday for RNA or protein direct introduction). In another embodiment, a PGCLC inducer in the form of DNA (e.g., viral or non-viral vectors) may be introduced into an epiblast or EpiLC in a form capable of being conditionally expressed in the cell. For example, when a drug (Tet)-responsive inducible vector is used, a method of maintaining expression by addition of tetracycline of a derivative thereof such as doxycycline to the medium during a desired period; when a vector having a transposon sequence is used, a method including, after lapse of a desired period, introducing transposase to remove the PGCLC inducer from the cell; and when a vector having LoxP sequences are used, a method including, after lapse of a desired period, introducing Cre recombinase to remove the PGCLC inducer from the cell and the like can be recited as examples. Instead of Tet-responsive inducible vector, other inducible vectors known in the art, such as a vector containing metallothionein promoter, may also be used.

When a PGCLC inducer in the form of DNA is introduced into an epiblast or EpiLC in a form capable of being conditionally expressed, the PGCLC inducer may also be introduced into the epiblast or EpiLC prior to the induction of the epiblast or EpiLC. For example, the PGCLC inducer can be introduced into a PSC before inducing differentiation into EpiLC. When the PSC as the starting material for EpiLC induction is an iPS cell, the PGCLC inducer can also be co-introduced into a somatic cell with reprogramming factors. Alternatively, when the PGCLC of interest is a non-human animal cell such as mouse, an epiblast can be isolated from an embryo of transgenic animal having the PGLC inducer in a form capable of being conditionally expressed in the epiblast. Likewise, a non-human animal PSC can be prepared from an appropriate cell source derived from the transgenic animal.

In view of clinical application to human therapy, it is preferable that the PGCLC inducer is not integrated into the genome of the epiblast or EpiLc nor stably maintained outside chromosome in order to avoid a risk of tumorigenesis. Therefore, in a preferable embodiment, the PGCLC inducer is in a form capable of disappearing from the PGCLC generated rapidly. As the PGCLC inducer for this purpose, a plasmid, RNA or protein may be exemplified. When the PGCLC inducer is in the form of DNA, non-integrated vector such as plasmid, adenoviral vector, Sendai viral vector, episomal vector may be used preferably. Since Sendai viral vector or episomal vector may be maintained outside chromosome for a relatively long period, it is more preferable to use a Sendai viral vector removable from PGCLC by siRNA or an episomal vector removable from PGCLC by Cre-loxP system. Alternatively, when the PGCLC inducer is introduced into an epiblast or EpiLC in a form of integrated vector such as retroviral vector, lentiviral vector and transposon, the vector is preferably designed such that it may be removed from chromosome of the PGCLC generated by Cre-loxP system of transposases.

The culture of epiblasts or EpiLCs into which the PGCLC inducer has been introduced for PGCLC induction may be performed as described in WO 2012/020687, Cell 146, 519-532 (2011) or Science 338, 971-975 (2012) except not adding cytokines including at least BMP4 in an amount effective for inducing epiblasts or EpiLCs into a PGC state (i.e., less than 100 ng/ml, preferably less than 50 ng/ml, more preferably less than 10 ng/ml). Most preferably, the medium lacks a detectable amount of BMP4.

As the basal medium for PGCLC induction, the basal media exemplified for the use in the EpiLC induction mentioned above (see Ib-2(ii)) are likewise preferably used. The medium may contain the same additives as those exemplified for the use in the EpiLC induction, as long as PGCLCs capable of contributing to normal spermatogenesis can be produced without going through transient mesodermal program by the method of the present invention.

The medium can be a serum-containing or serum-free medium (SFM). Preferably, a serum-free medium can be used. The concentration of serum (for example, fetal bovine serum (FBS), human serum, etc.) can be 0-20%, preferably 0-5%, more preferably 0-2%, most preferably 0% (i.e., serum-free). The SFM may contain or may not contain any alternatives to serum such as KSR.

The medium for PGCLC induction of the present invention can contain cytokines other than BMP4, including LIF, SCF, BMP8b and EGF, as long as PGCLCs capable of contributing to normal spermatogenesis can be produced without going through transient mesodermal program by the method of the present invention. Preferably, the medium contains no or a low amount of LIF (e.g., less than 300 U/ml, more preferably less than 100 U/ml). More preferably, the medium further contains no or low amounts of SCF and/or BMP8b and/or EGF (e.g., less than 30 ng/ml, more preferably less than 10 ng/ml for SCF; less than 100 ng/ml, more preferably less than 50 ng/ml for BMP8b; less than 10 ng/ml, more preferably less than 5 ng/ml for EGF). Most preferably, the medium lacks detectable amounts of BMP4, LIF, SCF, BMP8b and EGF.

In PGCLC induction culture of the present invention, epiblasts or EpiLCs are seeded to a cellular non-adhesive or low-adhesive culture vessel known per se to obtain a cell density of, for example, about 3 to 10×10⁴ cells/mL, preferably about 4 to 8×10⁴ cells/mL, and cultured in an incubator in an atmosphere of 1-10% CO₂/99-90% air at about 30-40° C., preferably about 37° C., for a period for expressing the introduced PGC inducer, for example, 1 to 10 days, preferably 1 to 6 days, more preferably 1 to 5 days, most preferably 2 to 4 days (e.g., 2, 3 or 4 days).

The fact of differentiation into PGCLCs can be confirmed by, for example, analyzing the expression of Blimp1 by RT-PCR and the like. As required, furthermore, the expression of other genes and cell surface antigens can also be examined. Examples of other genes include Stella. When epiblasts or EpiLCs (or embryos or PSCs as source thereof) bearing genes encoding fluorescent proteins under the control of Blimp1- and/or Stella-promoters are used as a starting material, the fact of differentiation into PGCLCs can be confirmed by FACS analysis. When the starting cells bear no appropriate transgenic reporter, such as ESCs or iPSCs derived from human, it is preferable to confirm the fact of differentiation into PGCLCs by FACS analysis and the like using one or more cell surface antigens specifically expressed on PGCLCs. As the cell surface antigens, preferably SSEA-1 and integrin-β3 are exemplified.

The method of producing PGCLCs from epiblasts isolated from embryos or EpiLCs induced from PSCs such as ES cells and iPS cells of the present invention is characterized in that it bypasses a mesodermal program that accompanies PGC specification in vivo and in vitro by cytokines including BMP4. As a result, according to the method of the present invention, PGCLCs can be obtained for a shorter period than that in the case using the cytokines. Importantly, the PGCLCs obtained by the method of the present invention robustly contribute to spermatogenesis and fertile offspring equal to or greater than the cytokine-induced PGCLCs, in spite of not going through a mesodermal program different from the PGC specification in vivo.

(II) Kit for Production of PGCLC from EpiLC

In another aspect, the present invention provides a kit for production of a PGCLC from an EpiLC. The kit can contain the aforementioned PGCLC inducer of the present invention, i.e., a PGCLC cell inducer containing the following TF(s):

(i) Blimp1, Prdm14 and Tfap2c;

(ii) Blimp1 and Prdm14;

(iii) Blimp1 and Tfap2c;

(iv) Prdm14 and Tfap2c;

(v) Prdm14; or

nucleic acid(s) encoding any of the TF(s) or (i) to (v) above (e.g., alcohol precipitate, frozen TE solution, lyophilizate and the like of a nucleic acid; lyophilizate of a protein, a frozen liquid dissolved in a suitable buffer, and the like), and the above-mentioned vector, cells, reagent and culture medium for introduction of said factor. This kit may further contain a protocol or instructions describing the step of induction into PGCLC.

In a preferable embodiment, the kit for production of a PGCLC from an epiblast or EpiLC of the present invention contains an isolated epiblast or EpiLC comprising nucleic acid(s) encoding exogenous transcription factor(s) selected from the group consisting of:

(i) Blimp1, Prdm14 and Tfap2c;

(ii) Blimp1 and Prdm14;

(iii) Blimp1 and Tfap2c;

(iv) Prdm14 and Tfap2c; and

(v) Prdm14;

wherein the nucleic acid(s) is/are in a form capable of being conditionally expressed in the epiblast or EpiLC. The kit can further contain a reagent for inducing the expression of the PGCLC inducer. As said reagent, doxycycline (Dox) for Tet-responsive inducible vectors, metal ion for vectors under the control of a metallothionein promoter may be exemplified.

In another preferable embodiment, the kit for production of a PGCLC from a PSC of the present invention contains an isolated PSC comprising nucleic acid(s) encoding exogenous transcription factor(s) selected from the group consisting of:

(i) Blimp1, Prdm14 and Tfap2c;

(ii) Blimp1 and Prdm14;

(iii) Blimp1 and Tfap2c;

(iv) Prdm14 and Tfap2c; and

(v) Prdm14;

wherein the nucleic acid(s) is/are in a form capable of being conditionally expressed in an EpiLC differentiated from the PSC. The kit can further contain a reagent for inducing the PSC into an EpiLC comprising ActA and optionally bFGF and/or KSR; and the above-mentioned reagent for inducing the expression of the PGCLC inducer.

In this context, the present invention also provides a method of producing a PGCLC from a PSC, which comprises the following steps I) and II):

I) the step for producing an EpiLC by culturing a PSC in the presence of ActA, optionally in the presence of further bFGF and/or KSR;

II) the step for inducing the EpiLC obtained in the step I) into a PGCLC by any of the methods described above. The method can further comprise the step III):

III) the step for selecting a Blimp1-positive cell from the cells obtained in the step II). As selection markers for FACS, surface antigens specific to PGCLC, SSEA-1, integrin-β3 and the like may also be used.

(III) Cell Population Containing PGCLCs Derived from Epiblasts or EpiLCs

The present invention also provides a cell population containing PGCLCs derived from epiblasts isolated from embryos or EpiLCs induced from PSCs, produced by the foregoing steps. The cell population may be a purified population of PGCLCs, and 1 kind or more of cells other than PGCLCs may be co-present. Here, “PGC-like cell (PGCLC)” is defined as a cell that shows elevated expression of Blimp1 and/or Stella compared to the EpiLC before inducing differentiation, is capable of contributing to normal spermatogenesis, and does not form teratoma when transplanted into an immunodeficient mouse. As stated above, when PGCLCs are induced using embryos or PSCs bearing genes encoding fluorescent proteins under the control of Blimp1- and/or Stella-promoters as a starting material, the Blimp1- and/or Stella-positive PGCLCs can be easily isolated and purified by sorting out the cell population obtained in the foregoing step using a cell sorter. The PGCLCs can also be isolated and purified by FACS using a reporter under the control of gene whose expression increases along with Blimp1 and Stella (e.g., Nanog) as a marker.

(IV) Use of PGCLCs Derived from Puluripotent Stem Cells

The PGCLCs derived from epiblasts isolated from embryos or EpiLCs induced from PSCs of the present invention can be used for varied purposes. For example, since the PGCLCs transplanted into a testis of a recipient animal can robustly contribute to spermatogenesis in the testis and the generation of healthy offspring, they can be used for the treatment of infertility or hereditary diseases of reproductive tissues.

The transplantation of the PGCLCs into a testis can be performed by using the PGCLCs in the same manner as described in WO 2012/020687 or Cell 146, 519-532 (2011).

The PGCLCs of the present invention can also be used for oogenesis (see Science 338, 971-975 (2012)).

The PGCLCs (including a cell population containing PGCLCs; the same applies below) of the present invention are produced as a parenteral preparation, preferably as an injection, suspension, or drip infusion, in a mixture with a pharmaceutically acceptable carrier, by a conventional means. Examples of the pharmaceutically acceptable carrier that can be contained in the parenteral preparation include aqueous liquids for injection, such as physiological saline and isotonic solutions containing glucose and other auxiliary drugs (e.g., D-sorbitol, D-mannitol, sodium chloride and the like). The agent of the present invention may be formulated with, for example, a buffering agent (e.g., phosphate buffer solution, sodium acetate buffer solution), a soothing agent (e.g., benzalkonium chloride, procaine hydrochloride and the like), a stabilizer (e.g., human serum albumin, polyethylene glycol and the like), a preservative, an anti-oxidant and the like.

When the agent of the present invention is prepared as an aqueous suspension, PGCLCs are suspended in one of the aforementioned aqueous liquids to obtain a cell density of about 1.0×10⁶ to about 1.0×10⁷ cells/ml.

The agent of the present invention can be cryopreserved under conditions typically used for the cryopreservation of stem cells, and thawed immediately before use.

Because the preparation thus obtained is stable and less toxic, it can be safely administered to mammals such as humans. Although the method of administration is not particularly limited, the preparation is preferably administered by injection or drip infusion into a seminiferous tuble when used for spermatogenesis. For a male infertility patient, for example, it is usually convenient to administer the agent in an amount of about 1.0×10⁵ to about 1×10⁷ cells, based on the amount of PGCLCs per dose, once or 2-10 times at about 1- to 2-week intervals. The present invention is hereinafter described in further detail by means of the following examples, to which, however, the invention is never limited.

EXAMPLES Methods

1. Animals

All animal experiments were conducted according to the Guidelines for Animal Experiments of Kyoto University. The BVSC transgenic mice (C57BL/6 background, Acc. No. BV, CDB0460T; SC CDB0465T: www.cdb.riken.jp/arg/TG%20mutant%20mice%20list.html) were established as reported previously (Reproduction 136, 503-514 (2008)). B6;129-Gt(ROSA)26Sor^(tm1(rtTA*M2)Jae)Col1a1^(tm2(tetO-Pou5f1)Jae)/J mice (Cell 121, 465-477 (2005)) (stock number: 006911) were purchased from the Jackson Laboratory. WBB6F1-W/W^(v) mice were purchased from SLC (Shizuoka, Japan).

2. Establishment of ESCs

Mice homozygous for the Rosa26-rtTA knock-in allele were obtained by crossing B6; 129-Gt(ROSA) 26Sor^(tm1(rtTA*M2)Jae)Col1a1^(tm2(tetO-Pou5f1)Jae)/J mice heterozygous for both loci. They were mated with BVSC transgenic mice and blastocysts were recovered at embryonic day (E) 2.5. BVSC-R26rtTA ESCs were selected by PCR genotyping, established and maintained under the N2B27 “2i+LIF” condition (Nature 453, 519-523 (2008)). A male cell line was used in this study.

3. Chimera Formation Assay

BVSC-R26rtTA ESCs were trypsinized and a single cell suspension was prepared. Approximately 15 ESCs/embryo were injected into blastocoels of E3.5 blastocysts obtained from ICR (albino) female mice with a piezo-actuating micromanipulator. Injected embryos were transferred into uteri of E2.5 pseudopregnant ICR female mice. Chimeric mice were delivered by Caesarean section at E18.5. Chimerism was determined by coat-colour. They were subjected to test breeding with ICR female mice to confirm the germline contribution.

4. Vector Construction

The mouse Blimp1 coding sequence (CDS) (from ATG in exon 3) and Tfap2c variant 1 (Accession number: NM_009335.2) CDS were cloned by PCR flanked with SalI-AviTag-XhoI and NotI sites and NotI and EcoRI sites, respectively. The Prdm14 CDS was obtained from AG-P14 (Cell Stem Cell 12, doi: 10.1016/j.stem.2012.12.012 (2013)).

The SalI-Kosak-Avi-Blimp1-NotI cassette was subcloned into XhoI/NotI sites of the pPyCAG-cHA-IP plasmid (Mol Cell Biol 22, 1526-1536 (2002)), and this cassette was subcloned again into the EcoRI/NotI sites of the pENTR1A Dual Selection Vector (Invitrogen). For Prdm14 and Tfap2c, KpnI-Kosak-3×FLAG-XhoI-G4S_Linker-SpeI and BamEI-Kosak-V5-G4S_Linker-NotI fragments, respectively, were attached to the N termini by PCR or synthesized oligonucleotide linker ligation. 3×FLAG-Prdm14 and V5-Tfap2c cassettes were subcloned into the KpnI/NotI and BamHI/EcoRI sites of pENTR1A, respectively. Lastly, they were shuttled into the PB-TET destination vector (Addgene) (Nature 458, 766-770 (2009)) with LR clonase II Enzyme Mix (Invitrogen). To construct pPBCAG-hph, a CAG promoter fragment from pCAGGS plasmid obtained by digestion of SpeI and EcoRI (filled) was inserted into the GG131 vector (Development 137, 3185-3192 (2010); Gene 108, 193-199 (1991)) digested with SpeI/MscI. All sequences engineered by PCR or oligonucleotide synthesis were confirmed. All attached sequences are shown in Table 1 and primer sequences for cloning are listed in Table 2.

TABLE 1  SalI-Kosak-AviTag-XhoI-(Blimp1 Exon3 ATG) GTCGACGCCACCATGTCCGGCCTGAACGACATCTTCGGCGCTCAGAAAAT CGAATGGCACGAACTCGAG-ATG) (SEQ ID NO: 13) KpnI-Kosak-3×FLAG-XhoI-G4S_Linker-SpeI- (Prdm14 ATG) GGTACCGCCACCATGGACTACAAGGACCACGACGGAGATTATAAGGATCA CGATATCGACTATAAGGATGACGACGATAAGCTCGAGTCTGGTGGCGGTG GCTCGGGCGGAGGTGGGTCGGGTGGCGGCGGATCAACTAGT-(ATG) (SEQ ID NO: 14) BamHI-Kosak-V5-G4S_Linker-NotI-(Tfap2c ATG) GGATCCGCCACCATGGGCAAGCCCATCCCTAACCCTCTGCTGGGCCTGGA CAGCACCTCTGGTGGCGGTGGCTCGGGCGGAGGTGGGTCGGGTGGCGGCG GATCAAGCGGCCGC-(ATG) (SEQ ID NO: 15)

TABLE 2  SalI-Kosak+A11+A2:B19+ GCGGTCGACGCCACCATGTCCGGCC A2:C21+A2:819 TGAACGACATCTTCGGCGCTCAGAA AATCGAATGGCACGAACTCGAGATG AAAATGGACATG (SEQ ID NO: 16) Blimp1-2568TAG-NotI_R2 GCGGGGCCGCCTAAGGATCCATCGG TTCAACTGTC (SEQ ID NO: 17) NotI_Tcfap2c_var1_F AAAGCGGCCGCATGTTGTGGAAAAT AAC (SEQ ID NO: 18) Tfap2c_var1_EcoRI_R ATAGAATTCTTACTTCCTGTGCTTT T (SEQ ID NO: 19) Avi-Blimp1_qF1 TGGTGCCTGTAAAGGTCAAAC (SEQ ID NO: 20) Avi-Blimp1_AttB2_qR1 GGCGGAATTAGCTTATCGAC (SEQ ID NO: 21) 3xFLAG-Prdm14_qF1 TCCTGGATCAAGAGGCTTTC (SEQ ID NO: 22) 3xFLAG-Prdm14_AttB2_qR2 ACTAGCTAGAGCGGCCATCAC (SEQ ID NO: 23) V5-Tfap2c_qF1 ATTCCAGCAAGACGATGGAG (SEQ ID NO: 24) V5-Tfap2c_AttB2_qR1 GGCGGAATTAGCTTATCGAC (SEQ ID NO: 25) b-geo_qF1 GCTTGCCGAATATCATGGTG (SEQ ID NO: 26) b-geo_qR1 CTTCAGCAATATCACGGGTAGC (SEQ ID NO: 27) Blimp1_CDS_qF1 GCCCACCTGCAGAAACACTAC (SEQ ID NO: 28) Blimp1_CDS_qR1 CCAGAATGCAATCGAAGGTG (SEQ ID NO: 29) Prdm14_CDS_qF1 CTTCCAGCCTGAACAAGCAC (SEQ ID NO: 30) Prdm14_CDS_qR1 GGAGTATGCTGGAGGCAGTG (SEQ ID NO: 31) Tfap2c_CDS_qF1 CCACGCGGAAGAGTATGTTG (SEQ ID NO: 32) Tfap2c_CDS_qR1 GTTGTTCCCGTTGGGTGTC (SEQ ID NO: 33) 5. Transfection and Selection of Subclones

BVSC-R26rtTA ESCs were transfected with PB-TET vectors containing key factors, pPBCAG-hph, and pCAGGS-mPB using Lipofectamine2000 (Invitrogen) on feeder cells (mouse embryonic fibroblasts) in a 60 mm dish under a “2i+LIF” condition. The total amount of vector DNA was below 8 μg. Transfectants were selected with Hygromycin B (150 μg/ml) (Sigma) and genotyped with PCR for transgenes. The primer sequences for the genotype are shown in Table 3.

TABLE 3  Genotype Avi-Blimp1_F3 AAATCGAATGGCACGAACTC (SEQ ID NO: 34) Avi-Blimp1_R2 GCATCCAGTTGCTTTTCTCC (SEQ ID NO: 35) 3xFLAG-Prdm14_F2 ATCGACTATAAGGATGACGAC (SEQ ID NO: 36) 3xFLAG-Prdm14_R1 GAGGTTCCTAAAGTGACTGTAG (SEQ ID NO: 37) V5-Tfap2c_F1 ATGGGCAAGCCCATCCCTAACCCT (SEQ ID NO: 38) V5-Tfap2c_R3 AAGGAGGCGGCTGGTACTCTGCAAC (SEQ ID NO: 39) Blimp1-mVenus_F ACTCATCTCAGAAGAGGATCTG (SEQ ID NO: 40) Blimp1-mVenus_R CACAGTCGAGGCTGATCTCG (SEQ ID NO: 41) Stella-ECFP_F CGAGCTAGCTTTTGAGGCTT (SEQ ID NO: 42) Stella-ECFP_R AACTTGTGGCCGTTTACGTC (SEQ ID NO: 43) Rosa-rtTA olMR8545_F AAAGTCGCTCTGAGTTGTTAT (SEQ ID NO: 44) olMR8546_R GGAGCGGGAGAAATGGATATG (SEQ ID NO: 45) olMR8052_R GCGAAGAGTTTGTCCTCAACC (SEQ ID NO: 46) 6. Southern Blotting

Eight micrograms of genomic DNA was isolated and digested with BamHI. DNA fragments were electrophoresed in 0.7% agarose gel, transferred to Hybond N+ (GE healthcare) and UV-crosslinked. The β-geo probe was obtained by digestion of PB-TET with RsrII/SmaI, labeled with ³²P (PerkinElmer) by a Random Primer DNA Labeling Kit Ver. 2.0 (TaKaRa) and purified with an Illustra ProbeQuant spin column (GE Healthcare). Radioisotope images were captured with a BAS system (Fujifilm).

7. TF- and Ck-PGCLCs

Transfected ESCs were adapted to a feeder-free condition prior to induction. EpiLC differentiation was performed as reported previously (Cell 146, 519-532 (2011)). After 36 hrs of differentiation, cells were harvested and cultured in a Lipidure-Coat 96-well plate (NOF) to be aggregated (started with 2,000 cells/well) in GK15 with 1.5 μg/ml of Dox (Clonetech). PGCLCs were induced by BMP4 (500 ng/ml), BMP8A (500 ng/ml), SCF (100 ng/ml), LIF (1000 U/ml) and EGF (50 ng/ml) as previously described (Cell 146, 519-532 (2011)). LDN193189 (120 pM; Stemgent) was added concurrently with Dox or Cks. Aggregates from ground-state ESCs were also cultured in GK15 with Dox as described above.

8. Reverse Transcription (RT) and Q-PCR

For evaluating endogenous transcripts, TF-induced BV-positive cells were FACS-sorted on d2 and d4 with the gates shown in FIG. 1c and FIG. 7b . The sorting gates used for d2 and d4 CK-PGCLCs are shown in FIG. 9a . Aggregates were trypsinized and lysed as a whole unless otherwise specified. Total RNA was purified with RNeasy micro (QIAGEN) and RT was performed with SuperScriptIII (Invitrogen) primed with oligo-dT primer according to the manufacturer's protocol. Real-time PCR was performed with Power SYBR (Applied Biosystems) and CFX384 (BioRad). The gene expression levels are presented as ΔCt (in log₂ scale) normalized (Mech Dev 113, 91-94 (2002)) with the average Ct values of Arbp and Ppia 14. To discriminate endogenous transcripts from exogenous ones, both the oligo-dT primer (Invitrogen) and gene-specific primers of interest were used for RT to reduce the RT bias due to differences in the distance between RT priming sites and amplified regions. The amplification efficiency of the newly designed primer sets was determined with pGEM-T-Easy plasmids harbouring the corresponding amplicons as templates. To verify both the endogenous and exogenous expression levels, samples were tested with CDS primers concurrently (data not shown). The primer sequences are listed in Table 4 (Biol Reprod 75, 705-716 (2006); Nucleic Acids Res 34, e42 (2006)).

TABLE 4  Quantitative RT-PCR Blimp1_F AGCATGACCTGACATTGACACC (SEQ ID NO: 47) Blimp1_R CTCAACACTCTCATGTAAGAGGC (SEQ ID NO: 48) Prdm14_F ACAGCCAAGCAATTTGCACTAC (SEQ ID NO: 49) Prdm14_R TTACCTGGCATTTTCATTGCTC (SEQ ID NO: 50) Tfap2c_F GGGCTTTTCTCTCTTGGCTGGT (SEQ ID NO: 51) Tfap2c_R TCCACACGTCACCCACACAA (SEQ ID NO: 52) Nanos3 F CACTACGGCCTAGGAGCTTGG (SEQ ID NO: 53) Nanos3_R TGATCGCTGACAAGACTGTGGC (SEQ ID NO: 54) stella_F AGGCTCGAAGGAAATGAGTTTG (SEQ ID NO: 55) stella_R TCCTAATTCTTCCCGATTTTCG (SEQ ID NO: 56) Pou5f1_F GATGCTGTGAGCCAAGGCAAG (SEQ ID NO: 57) Pou5f1_R GGCTCCTGATCAACAGCATCAC (SEQ ID NO: 58) Sox2_F CATGAGAGCAAGTACTGGCAAG (SEQ ID NO: 59) Sox2_R CCAACGATATCAACCTGCATGG (SEQ ID NO: 60) Nanog_F CTTTCACCTATTAAGGTGCTTGC (SEQ ID NO: 61) Nanog_R TGGCATCGGTTCATCATGGTAC (SEQ ID NO: 62) Dnmt3a_F GACTCGCGTGCAATAACCTTAG (SEQ ID NO: 63) Dnmt3a_R GGTCACTTTCCCTCACTCTGG (SEQ ID NO: 64) Dnmt3b_F CTCGCAAGGTGTGGGCTTTTGTAAC (SEQ ID NO: 65) Dnmt3b_R CTGGGCATCTGTCATCTTTGCACC (SEQ ID NO: 66) Hoxa1_F GTGACTAGTCTTCTGCATGTCG (SEQ ID NO: 67) Hoxa1_R TCTGCTCTGGACCACATCACTC (SEQ ID NO: 68) Hoxb1_F GATCCTACAGGTCTTGGGACC (SEQ ID NO: 69) Hoxb1_R AGCTCAAAGGCACTGAACTGAG (SEQ ID NO: 70) T_F ATCAGAGTCCTTTGCTAGGTAG (SEQ ID NO: 71) T_R GTTACAATCTTCTGGCTATGC (SEQ ID NO: 72) Gata6_F CACAGTCCCCGTTCTTTTACTG (SEQ ID NO: 73) Gata6_R GTGGTACAGGCGTCAAGAGTG (SEQ ID NO: 74) Arbp_F CAAAGCTGAAGCAAAGGAAGAG (SEQ ID NO: 75) Arbp_R AATTAAGCAGGCTGACTTGGTTG (SEQ ID NO: 76) Ppia_F TTACCCATCAAACCATTCCTTCTG (SEQ ID NO: 77) Ppia_R AACCCAAAGAACTTCAGTGAGAGC (SEQ ID NO: 78) 9. LacZ Staining

Cell aggregates at 12 hrs were trypsinized and fixed with 2% paraformaldehyde and 0.2% glutaraldehyde. Fixed cells were spread with Cytospin4 (Thermo Scientific) and stained with LacZ staining solution overnight (Nature 458, 766-770 (2009)).

10. Flow Cytometric Analysis and Cell Sorting

The sample preparations from cell aggregates were performed essentially as described previously (Cell 146, 519-532 (2011)). Fluorescent-activated cell sorting (FACS) was performed with a FACSAria or FACSAriaIII (BD) cell sorter. BV and SC fluorescence was detected with FITC and AmCyan Horizon V500 channel, respectively. Data were analyzed with FACSDiva (BD) or Flowjo (Tree Star Inc.) software.

11. Immunofluorescent Staining

BV-positive cells from BP14A-induced d4 aggregates were sorted with the gate shown in FIG. 9c , mixed with EpiLCs at a ratio of 1:1 and spread onto MAS-coated glass slides. Immunofluorescent staining was performed as reported previously (Cell 146, 519-532 (2011)). The primary antibodies (Abs) used in this study were as follows: anti-GFP (rat, monoclonal Ab (MAb); Nacalai Tesque), anti-DNMT3B (mouse, MAb; Imgenex), anti-H3K27me3 (rabbit, polyclonal Ab (pAb); Millipore), and anti-H3K9me2 (rabbit, pAb). Secondary antibodies were as follows: Alexa Fluor 568 anti-rabbit IgG, Alexa Fluor 488 anti-rat IgG, and Alexa Fluor 647 anti-mouse IgG (all three from Invitrogen). Images were captured with a confocal laser scan microscope (Olympus FV1000).

12. Bisulfite Sequencing

Genomic DNA was isolated and bisulfite treatment was conducted with an EpiTect Bisulfite Kit (QIAGEN) according to the manufacturer's protocol. The differentially methylated regions of Snrpn and H19 were amplified by PCR as previously reported (Genomics 79, 530-538). Sequences were determined and analyzed with QUMA (http://quma.cdb.riken.jp/top/index.html) (Nucleic Acids Res 36, W170-175 (2008)).

13. cDNA Amplification and Microarray Analysis

The cells surrounded with red rectangles in FIG. 9a were sorted by FACS. Note that the background level was different in sorting of CK-PGCLCs and TF-PGCLCs in D2. Total RNA isolation, reverse transcription and cDNA amplification were conducted as previously described (Cell 146, 519-532 (2011); Nucleic Acids Res 34, e42 (2006)). Samples were analyzed with a GeneChip Mouse Genome 430_2.0 Array (Affimetrix). Data were normalized with dChip and are shown in log 2 scale (Proc Natl Acad Sci USA 98, 31-36 (2001)). Probe selection criteria for analysis were as follows: (1) maximum expression score≥8, (2) maximum differential expression level≥2, (3) the highest average expression level among multiple probes for a gene, if any. Published data (GSE30056 (GSM744095-GSM744096, GSM744101-GSM744104)) were included in the analysis (Cell 146, 519-532 (2011)). We selected 4,479 probes and performed principal component analysis (PCA) with R (version 2.15.1) (R_Development_Core_Team. R, R Foundatin for Statistical Computing, 2012). For the differential gene expression analysis, we averaged biological duplicates or quadruplicates and selected “core PGC genes” (FIG. 9b , upper) and “somatic mesodermal genes” (Supplementary FIG. 9b , lower) according to the following criteria. “core PGC genes” were i) upregulated in BP14A-induced d2 more than 4-fold compared with both EpiLCs and the parental clone with Dox d2; ii) not downregulated in E9.5 PGCs (the differential expression level was less than 2-fold greater compared with that of BP14A-induced d2). “somatic mesodermal genes” exhibited at least 4-fold up-regulation in PGCLC d2 as compared with both EpiLCs and BP14A-induced d2. Representative genes are specified.

14. Seminiferous Tubule Injection

After the designated cell populations were sorted by FACS, 1×10⁴ cells/testis were injected into the neonatal testes of W/W^(v) mice (7 dpp) basically as previously described (Development 132, 117122 (2005)). Anti-mouse CD4 antibody (50 mg/dose, clone GK1.5; eBioscience or Biolegend) was injected intraperitoneally at day 0, 2, or 4 for immunosuppression as necessary (Biol Reprod 68, 167-173 (2003)). The transplanted testes were analyzed 10 weeks after injection. For hematoxylin and eosin staining, testis samples were fixed with Bouin's solution, embedded in paraffin, and sectioned.

15. Intracytoplasmic Sperm Injection (ICSI)

ICSI was performed basically as reported previously (Biol Reprod 52, 709-720 (1995)). Briefly, seminiferous tubules with speimatogenesis colonies were gently minced and a spermatogenic cell suspension was prepared. Spermatozoa were injected into oocytes recovered from BDF1 mice. After in vitro embryo culture, 2-cell-stage embryos were transferred into the oviducts of E0.5 pseudopregnant mice (ICR). Pups were delivered by Caesarean section at E18.5. The primer sequences used for genotyping PCR are described in Table 3.

Results

We derived ESCs expressing mVenus and ECFP under the control of Blimp1 and stella (also known as Dppa3/Pgc7) regulatory elements (BVSC), respectively (Reproduction 136, 503-514 (2008)), and reverse tetracycline transactivator (rtTA) under the control of the constitutively active Rosa26 locus (Cell 121, 465-477 (2005)) (BVSCR26rtTA ESCs) (FIG. 1a ). During mouse development, Blimp1 expression signifies the onset of PGC specification, whereas stella begins expression in the established PGCs, and the BVSC expression is a faithful indicator for PGC specification and development both in vivo and in vitro (Cell 146, 519-532 (2011); Science 338, 971-975 (2012); Reproduction 136, 503-514 (2008)). We constructed piggyback transposon-based vectors expressing Blimp1, Prdm14 or Tfap2c under the control of tetracycline regulatory elements (TREs) and infected the BVSCR26rtTA ESCs karyotype) (FIG. 5a-c ) with these vectors to isolate BVSCR26rtTA ESCs bearing transgenes for all three TFs (BVSCR26rtTA BP14A cells), two of the three TFs (BVSCR26rtTA BP14, BA and P14A cells), or one of the three TFs (BVSCR26rtTA B, P14, and A cells) (FIGS. 1a, 5d and 5e ).

We first examined whether simultaneous forced expression of the three TFs induces EpiLCs into the germ cell fate. We induced BVSCR26rtTA BP14A cells (hereafter BP14A cells, Line 3-3) into EpiLCs, and then generated floating aggregates of ˜2,000 EpiLCs in the absence of relevant cytokines with or without doxycycline (Dox, 1.5 μg/ml), a tetracycline analogue (FIG. 6a ). The floating aggregates of EpiLCs without Dox did not show BVSC expression over the six-day period (FIG. 1b ). In contrast, remarkably, those with Dox exhibited robust BVSC expression as early as day 2 (d2) of the Dox treatment (FIG. 1b ). We confirmed that Dox induces exogenous TFs rapidly and at a high level in nearly all EpiLCs (FIG. 6b, c ).

Fluorescence activated cell sorting (FACS) revealed that at d2 of Dox treatment, more than ˜80% and ˜30% of the cells expressed BV and SC, respectively (FIG. 1c ), and the efficiency of BVSC induction was dependent on the dosage of Dox (FIG. 6d, e ). Notably, the BVSC induction by Dox was much more efficient and faster than that by the cytokines: Most typically, the cytokines (BMP4, LIF, BMP8A, SCF, EGF) induce strong BV and SC around day 2 and 4, respectively (Cell 146, 519-532 (2011); Science 338, 971-975 (2012)) (FIG. 6f ).

The BVSC-positive cells induced by Dox showed proliferation and persisted until day 4, but decreased thereafter (FIG. 1b ). The other BP14A lines (Lines 3-6, 3-10) showed similar BVSC induction by Dox (FIG. 7a, c ).

These findings demonstrate that the three TFs, BLIMP1, PRDM14 and TFAP2C, induced by Dox are sufficient for rapid and robust activation of the BVSC transgenes in EpiLCs.

We next set out to examine the expression of genes relevant for PGC specification in TF (BP14A)-induced BV-positive cells during the course of 96 hrs by Q-PCR.

We first confirmed that robust induction of exogenous TFs precedes endogenous Blimp1, Prdm14, and Tfap2c activation in induced whole-cell aggregates (FIG. 8a, b ). We found that, as in the case of cytokine-induced BV-positive cells, TF-induced BV-positive cells showed up-regulation of key genes for PGC specification (Blimp1, Prdm14, Tfap2c, Nanos3, stella, Pou5f1, Sox2, Nanog) and down-regulation of key epigenetic modifiers (Dnmt3a and Dnmt3b) (FIG. 1d ). Interestingly, we noted that in contrast to cytokine-induced BV-positive cells that exhibited transient up-regulation followed by repression of key mesodermal genes such as Hoxa1, Hoxb1 and T (Brachyury) (Cell 146, 519-532 (2011)), which was also seen during PGC specification in vivo (Nature 418, 293-300 (2002); Biol Reprod 75, 705-716 (2006); Genes Dev 22, 1617-1635 (2008)), TF-induced BV-positive cells did not show transient up-regulation of these genes, but rather continued to express them at low levels, or not at all (FIG. 1d ).

These findings suggest that the TF (BP14A)-induced BV-positive cells acquire a transcriptional program similar to PGCs, but they lack transient acquisition of a mesodermal program, which is evident both during PGC specification in vivo and PGCLC induction by cytokines (see below).

We next analyzed whether forced expression of two of the three TFs or forced expression of one of the three TFs would induce BVSC in floating aggregates of EpiLCs (We evaluated at least three independent lines for each combination of TFs and for each TF).

We found that P14A, and to a lesser extent, BP14 and BA, and, strikingly, Prdm14 alone, activated BVSC, although all at lower efficiencies compared to the three TFs (FIG. 7a-c ). We noted that the BA-induced aggregates (two out of three lines) looked somewhat fragile and remained small (FIG. 7a ), and that forced expression of Blimp1 or Tfap2c alone did not activate BVSC (FIG. 7a-c ).

We confirmed that all the lines show essentially uniform induction of exogenous TF(s) upon Dox treatment (FIG. 8c-g ). It should be noted that we were not able to isolate BVSCR26rtTA B cells that express exogenous Blimp1 upon Dox treatment at levels as high as those of exogenous Prdm14 in BVSCR26rtTA P14 cells (FIG. 8d ).

The two TFs (P14A, BP14, BA)- and single TF (Prdm14)-induced BV-positive cells exhibited gene expression dynamics very similar to those of the three TF (BP14A)-induced BV-positive cells (FIG. 7d ), suggesting that once the key transcriptional circuitry for PGC specification is activated, the induced cells acquire similar transcriptional profiles.

We then determined the relationship between the rate of BVSC induction and the level of exogenous TF expression. As shown in FIG. 1e (data based on FIGS. 7c and 8d ), BP14A induced BVSC much more efficiently than P14A or BA or P14 at similar whole exogenous TF transcript levels, indicating that Blimp1, Prdm14, and Tfap2c show a synergistic effect on the activation of PGC-like transcriptional profiles in EpiLCs.

To exclude the possibility that the TFs activate cytokine signaling, particularly BMP4 signaling, which in turn induces EpiLCs into a PGC-like state, we induced BP14A in EpiLCs with or without an inhibitor for BMP4 signaling [LDN193189, an inhibitor for activin receptor-like kinase 2/3 (ALK2/3)].

As shown in FIG. 2a , while LDN193189 efficiently blocked BV induction in EpiLCs by BMP4, it had no effect on BVSC induction by BP14A. Q-PCR analysis confirmed that LDN193189 inhibited Blimp1 and Prdm14 induction by BMP4 but not that by BP14A (FIG. 2b ), demonstrating that induction of a PGC-like state by TFs results from direct activation of a PGC program by TFs.

We then examined whether induction of a PGC-like state by TFs requires an EpiLC state as an epigenetic background. While BP14A induced EpiLCs robustly into a PGC-like state, BP14A induction in ESCs resulted in a somewhat peculiar phenotype: intense SC activation with no BV (FIG. 2c, d ).

We therefore conclude that a proper epigenetic background is essential for robust induction of a PGC-like state by TFs.

To characterize more fully the properties of the TF-induced PGC-like cells (hereafter referred to as TF-PGCLCs), we next determined the global transcription profiles of TF-PGCLCs [BV-positive cells induced by BP14A (d2 and d4), BP14 (d2), P14A (d2), P14 (d2)] by microarray analysis and compared them with those of PGCs in vivo [PGCs at embryonic day (E) 9.5] and cytokine-induced d2, d4, and d6 PGCLCs (hereafter referred to as Ck-PGCLCs) (FIG. 9a ) (Cell 146, 519-532 (2011)).

Principal component analysis (PCA) revealed that all the TF-PGCLCs, irrespective of the TF combinations or of the induction period, bear similar transcriptomes, which are also similar to the transcriptomes of d4 and d6 Ck-PGCLCs and, to a lesser extent, of E9.5 PGCs (FIG. 3a ), corroborating the idea that the exogenous TFs, at varying efficiencies depending on their combinations, activate endogenous key transcription circuitry for PGC specification, which creates a similar PGC-like state.

Consistent with the requirement of an appropriate epigenome for the TF-induced PGC-like state (FIG. 2c, d ), the transcriptomes of the SC-positive cells induced by BP14A in ESCs were clearly different from those of TF/Ck-PGCLCs and PGCs, and were closer to those of ESCs (FIG. 3a ). Notably, d2 Ck-PGCLCs showed a considerably different global transcription profile from those of TF-PGCLCs, d4 and d6 Ck-PGCLCs, and E9.5 PGCs, but a more similar profile to that of E5.75 epiblasts, indicating that d2 Ck-PGCLCs represent a transient state toward the acquisition of a PGC-like state from the EpiLC/epiblast states (FIG. 3a ).

We looked at individual genes up-regulated in d2 TF (BP14A)-PGCLCs in comparison to those in EpiLCs/control EpiLCs without exogenous TFs but treated with Dox and found that genes such as Blimp1 (endogenous), Prdm14 (endogenous), Tfap2c (endogenous), stella, Sox2, K1f2, Tcl1, Esrrb, E1f3, Kit, Lifr, Nr5a2, Gjb3, Tdh, Spnb3, Pyg1, Mbp, Mtap7, Npnt, and AU015386 showed robust up-regulation: All these genes (“core PGC genes”) were also up-regulated in d4 and d6 Ck-PGCLCs and in E9.5 PGCs (FIG. 9b ).

We then examined the genes that showed up-regulation in d2 Ck-PGCLCs but not in d2 TF-PGCLCs in comparison to those in EpiLCs and this analysis revealed that the genes Hoxa1, Hoxb1, Hoxb2, Evx1, T(Brachyury), Cdx1, Cdx2, Hand1, Snai1, Mesp1, Id1, Msx1, Msx2, Nkx1.2, Isl1, Mixl1, Rspo3, Wnt5a, Fgf8 and Bmp4, all of which (“somatic mesodermal genes”) show transient up-regulation in PGC precursors at around E6.75 to E7.25 and represent a somatic mesodermal program (Genes Dev 22, 1617-1635 (2008)), were transiently up-regulated in d2 Ck-PGCLCs but not in d2 TF-PGCLCs, and these genes were also down-regulated in d4 and d6 Ck-PGCLCs as well as in E9.5 PGCs (FIG. 9b ).

Collectively, these findings provide evidence on a genome-wide scale that PGC specification by BMP4 activates both a key PGC program and somatic mesodermal program, the latter of which is eventually repressed by the former, and that the direct activation of key TFs confers EpiLCs with the key PGC program but not the somatic mesodermal program.

We next evaluated the epigenetic profiles of TF-PGCLCs. Immunofluorescence analysis revealed that, compared to EpiLCs that were exclusively positive for DNMT3B, BV-positive d4 TF-PGCLCs were negative/extremely weak for DNMT3B and showed a reduced level of Histone H3 Lysine 9 di-methylation (H3K9me2) and an elevated level of H3K27 tri-methylation (H3K27me3) (FIG. 3b , FIG. 9c ). Analysis of the imprinting state by bisulfite sequencing showed that BV-positive d4 TF-PGCLCs retained the imprints on the paternally imprinted H19 and on the maternally imprinted Snrpn (FIG. 3c ).

These findings suggest that the BV-positive d4 TF-PGCLCs acquire an epigenome similar to d6 Ck-PGCLCs and to migrating PGCs at E8.5-E9.5 (Cell 146, 519-532 (2011); Development 134, 2627-2638 (2007)).

Upon Dox withdrawal, the TF-PGCLCs should shut off exogenous TFs, but continue their endogenous transcription program, and may therefore serve as precursors for proper spermatogenesis.

To explore this possibility, we induced TF-PGCLCs by BP14A, purified the BV-positive cells (at d3, d4, and d6) (FIG. 4a ), and transplanted them into seminiferous tubules of neonatal W/W^(v) mice that lacked endogenous germ cells (Cell 146, 519-532 (2011); Development 132, 117-122 (2005)). Ten weeks after transplantation, we isolated the transplanted testes and examined for the presence of the seminiferous tubules with spermatogenesis. Strikingly, the testes transplanted with the TF-PGCLCs, particularly those sorted at d3 and d4, contained numerous tubules with signs of spermatogenesis (FIG. 4b, k ): These tubules indeed contained abundant spermatozoa with proper morphology (FIG. 4c, d ).

Histological examination revealed that the spermatogenesis originated by TF-PGCLCs proceeded in a normal fashion (FIG. 4c ). The control Ck-PGCLCs also contributed to proper spermatogenesis (FIG. 4k ). In contrast, the SC-positive cells induced by activation of BP14A in ESCs did not contribute to spermatogenesis, but instead formed many foci of teratomas in six out of eight transplanted testes (FIG. 4k , FIG. 10a, b ). We fertilized wild-type oocytes with TF-PGCLC-derived spermatozoa by intracytoplasmic sperm injection (ICSI) (Biol Reprod 52, 709-720 (1995)). The resultant zygotes developed in an apparently normal fashion into 2-cell embryos (FIG. 4e, f , FIG. 10c ). We transferred these embryos into oviducts of foster mothers and, 19 days later, obtained healthy offspring with grossly normal placenta (FIG. 4g, h ). These offspring indeed bore transgenes for the exogenous TFs and the BVSCR26rtTA (FIG. 4i ), but nonetheless grew up normally into fertile adults (FIG. 4j , FIG. 10d, e ).

We conclude that the TF-PGCLCs function as bona fide precursors for the spermatogenesis and healthy offspring.

We have demonstrated that the three TFs examined herein, BLIMP1, PRDM14, and TFAP2C, activate a key PGC program in a synergistic fashion on an appropriate epigenetic background. The synergistic action of the three TFs indicates their mutual regulation. We have also shown that PRDM14 is a minimum requirement for this activation and that neither BLIMP1 alone nor TFAP2C alone is sufficient to activate the PGC program by itself.

The system we have presented not only offers an opportunity to clarify the precise mechanism of epigenetic reprogramming in PGCs (genome-wide DNA demethylation and histone modification changes (Development 139, 15-31 (2012)), but also points to the importance of conducting mechanistic studies to determine how the key TFs work on an appropriate epigenetic background. It should also be feasible to explore TF-based regulation of further critical processes of germ cell development. The TF-based control of germ cell development may also be applicable to mammals other than mice, including humans.

While the present invention has been described with emphasis on preferred embodiments, it is obvious to those skilled in the art that the preferred embodiments can be modified. The present invention intends that the present invention can be embodied by methods other than those described in detail in the present specification. Accordingly, the present invention encompasses all modifications encompassed in the gist and scope of the appended “CLAIMS.”

In addition, the contents disclosed in any publication cited herein, including patents and patent applications, are hereby incorporated in their entireties by reference, to the extent that they have been disclosed herein.

This application is based on patent application No. 61/771,619 filed in U.S.A., the contents of which are hereby incorporated by reference. 

The invention claimed is:
 1. A method of producing a primordial germ cell-like cell (PGCLC) from an isolated epiblast or epiblast-like cell (EpiLC), which comprises allowing the epiblast or EpiLC to express exogenous transcription factor(s) selected from the group consisting of: (i) Blimp1, Prdm14 and Tfap2c; (ii) Blimp1 and Prdm14; (iii) Blimp1 and Tfap2c; (iv) Prdm14 and Tfap2c; and (v) Prdm14; thereby inducing the epiblast or EpiLC into a PGC state without acquiring transient mesodermal program, wherein the epiblast or EpiLC is induced into a PGCLC in the absence of BMP4.
 2. The method according to claim 1, wherein nucleic acid(s) encoding the exogenous transcription factor(s) is/are introduced into the epiblast or EpiLC.
 3. The method according to claim 1, wherein nucleic acid(s) encoding the exogenous transcription factor(s) has/have been introduced into the epiblast or EpiLC, in a form capable of being conditionally expressed, prior to the induction of the epiblast or EpiLC.
 4. The method according to claim 3, wherein the epiblast or EpiLC is cultured under conditions which the nucleic acid(s) encoding the exogenous transcription factor(s) is/are expressed for 1 to 5 days.
 5. The method according to claim 1, wherein the EpiLC is obtained by culturing a pluripotent stem cell (PSC) in the presence of activin A (ActA), optionally in the presence of further basic fibroblast growth factor (bFGF) and/or Knockout™ Serum Replacement (KSR).
 6. The method according to claim 5, wherein the PSC is an embryonic stem cell (ESC) or induced pluripotent stem cell (iPSC).
 7. The method according to claim 1, wherein nucleic acid(s) encoding the exogenous transcription factor(s) is/are in a form capable of disappearing from the PGCLC.
 8. The method according to claim 7, wherein the nucleic acid(s) is/are carried on vector(s) selected from the group consisting of plasmid, episomal vector, transposon, adenoviral vector and Sendai viral vector.
 9. The method according to claim 1, wherein the EpiLC is derived from mouse or human.
 10. A method of producing a PGCLC from a PSC, which comprises the following steps I) and II): I) the step for producing an EpiLC by culturing a PSC in the presence of ActA, optionally in the presence of further bFGF and/or KSR; II) the step for inducing the EpiLC obtained in the step I) into a PGCLC by the method according to claim
 1. 11. The method according to claim 10, which further comprises: III) the step for selecting a Blimp1-positive cell from the cells obtained in the step II).
 12. The method according to claim 1, wherein the epiblast or EpiLC is induced into a PGCLC in the absence of BMP4, LIF, SCF, BMP8b, and EGF.
 13. The method according to claim 10, wherein the step II) is performed in the absence of BMP4, LIF, SCF, BMP8b, and EGF.
 14. The method according to claim 1, wherein exogenous transcription factor(s) is/are selected from the group consisting of: (i) Blimp1, Prdm14 and Tfap2c; (iii) Blimp1 and Tfap2c; (iv) Prdm14 and Tfap2c; and (v) Prdm14.
 15. The method according to claim 1, wherein exogenous transcription factor(s) is/are selected from the group consisting of: (i) Blimp1, Prdm14 and Tfap2c; (iii) Blimp1 and Tfap2c; and (iv) Prdm14 and Tfap2c.
 16. The method according to claim 10, wherein exogenous transcription factor(s) is/are selected from the group consisting of: (i) Blimp1, Prdm14 and Tfap2c; (iii) Blimp1 and Tfap2c; (iv) Prdm14 and Tfap2c; and (v) Prdm14.
 17. The method according to claim 10, wherein exogenous transcription factor(s) is/are selected from the group consisting of: (i) Blimp1, Prdm14 and Tfap2c; (iii) Blimp1 and Tfap2c; and (iv) Prdm14 and Tfap2c. 